Compositions and methods for prevention and treatment of amyloid-β peptide-related disorders

ABSTRACT

The present invention provides methods and compositions for modulating levels of amyloid-β peptide (Aβ) exhibited by cells or tissues. The invention also provides pharmaceutical compositions and methods of screening for compounds that modulate Aβ levels. The invention also provides modulation of Aβ levels via selective modulation (e.g., inhibition) of ATP-dependent γ-secretase activity. The invention also provides methods of preventing, treating or ameliorating the symptoms of a disorder, including but not limited to an Aβ-related disorder, by administering a modulator of γ-secretase, including, but not limited to, a selective inhibitor of ATP-dependent γ-secretase activity or an agent that decreases the formation of active (or optimally active) γ-secretase. The invention also provides the use of inhibitors of ATP-dependent γ-secretase activity to prevent, treat or ameliorate the symptoms of Alzheimer&#39;s disease.

RELATED APPLICATIONS

This application claims benefit, under 35 U.S.C. §119(e), of U.S.provisional application No. 60/345,009, filed on Jan. 4, 2002, which isincorporated herein by reference in its entirety.

This invention was made with Government support under grant number5P01AG009464-07 awarded by the National Institutes of Health. The UnitedStates Government may have certain rights in the invention.

1. TECHNICAL FIELD

The present invention relates to methods and compositions for modulatinglevels of amyloid-β peptide (Aβ) exhibited by cells or tissues. Theinvention also relates to pharmaceutical compositions and methods ofscreening for compounds that modulate Aβ levels. The invention alsorelates to modulation of Aβ levels via selective modulation (e.g.,inhibition) of ATP-dependent γ-secretase activity. The invention alsorelates to methods of preventing, treating or ameliorating the symptomsof a disorder, including but not limited to an Aβ-related disorder, byadministering a modulator of γ-secretase, including, but not limited to,a selective inhibitor of ATP-dependent γ-secretase activity or an agentthat decreases the formation of active (or optimally active)γ-secretase. The invention also relates to the use of inhibitors ofATP-dependent γ-secretase activity to prevent, treat or ameliorate thesymptoms of Alzheimer's disease.

2. BACKGROUND OF THE INVENTION

Amyloid-β (Aβ) peptides are metabolites of the Alzheimer'sdisease-associated precursor protein, β-amyloid precursor protein (APP),and are believed to be the major pathological determinants ofAlzheimer's disease (AD). These peptides consist mainly of 40 to 42amino acids, Aβ1-40 (“Aβ40”) and Aβ1-42 (“Aβ42”), respectively. Aβ40 andAβ42 are generated by two enzymatic cleavages occurring close to theC-terminus of APP. The enzymes responsible for the cleavage, β-secretaseand γ-secretase, generate the − and C-termini of Aβ, respectively. Theamino terminus of Aβ is formed by β-secretase cleavage betweenmethionine residue 596 and aspartate residue 597 of APP (APP 695 isoformnumbering) (see, e.g., U.S. Pat. No. 6,440,698; and U.S. Pat. No.5,744,346).

γ-secretase activity cleaves at varying positions 38-, 40- or43-residues C-terminal of this β-secretase cleavage to release Aβpeptides (for a review of γ-secretase and γ-secretase activity, see,e.g., U.S. Patent Application 20020025540). The complete molecularidentity of γ-secretase enzyme is still unknown. Presenilin 1, or theclosely related presenilin 2, is needed for γ-secretase activity.γ-secretase activity is reduced 80% in cultured cells derived fromembryos genetically deleted for presenilin 1. All γ-secretase activityis lost in cells lacking both presenilin 1 and presenilin 2.Peptidomimetic inhibitors of γ-secretase activity can be crosslinked topresenilins 1 and 2, suggesting that these proteins are catalyticsubunits for the cleavage. However, γ-secretase activity isolated fromcells chromatographs as a large complex>1 M daltons. Recent geneticstudies have identified three more proteins required for γ-secretaseactivity; nicastrin, aph-1 and pen-1. (Francis et al., 2002,Developmental Cell 3(1): 85-97; Steiner et al., 2002, J. Biol.Chemistry: 277(42): 39062-39065; and Li et al., 2002, J. Neurochem.82(6): 1540-1548). Accumulation of presenilin into high molecular weightcomplexes is altered in cells lacking these proteins.

A third enzyme, α-secretase, cleaves the precursor protein between theβ- and γ-cleavage sites, thus precluding Aβ production and releasing anapproximately 3 kDa peptide known as P3, which is non-pathological. Bothβ- and α-secretase cleavage also result in soluble, secreted—terminalfragments of APP, known as sAPβ and sAPPα, respectively. The sAPPαfragment has been suggested to be neuroprotective.

In normal individuals, the Aβ peptide is found in two predominant forms,the majority Aβ-40 (also known as Aβ1-40) form and the minority Aβ42(also known as Aβ1-42) form, each having a distinct COOH-terminus. Themajor histological lesions of AD are neuritic plaques andneurofibrillary tangles occurring in affected brain regions. Neuriticplaques consist of Aβ peptides, primarily Aβ40 and Aβ42. Althoughhealthy neurons produce at least ten times more Aβ40 compared to Aβ42,plaques contain a larger proportion of the less soluble Aβ42. Patientswith the most common form of familial Alzheimer's disease show anincrease in the amount of the Aβ42 form. The Aβ40 form is not associatedwith early deposits of amyloid plaques. In contrast, the Aβ42 formaccumulates early and predominantly in the parenchymal plaques and thereis strong evidence that Aβ42 plays a major role in amyloid plaquedeposits in familial Alzheimer's disease patients (Roher et al., 1993,Proc. Natl. Acad. Sci. USA 90:10836; Iwatasubo, T., et al., 1994 Neuron13:45; Yamaguchi et al, 1995, Amyloid Int. J. Clin. Invest. 2:7-16; andMann et al., 1996 Am. J. Pathol. 148:1257).

Neurofibrillary tangles consist of aggregated tau protein and their rolein AD pathology is less clear. AD symptoms are most closely correlatedwith total brain Aβ rather than plaques. About 10% of AD cases resultfrom autosomal dominant inheritance of mutations in either the APP orthe presenilin 1 and presenilin 2 genes. In both cases, increasedproduction of total Aβ or Aβ42 versus Aβ40 results. There is strongevidence that Aβ peptides are critical in the pathogenesis ofAlzheimer's disease. Therefore, compositions which modulate the activityand/or levels of these peptides are desired.

For example, U.S. Patent Application 20020128319 A1 states that certainnon-steroidal anti-inflammatory drugs (NSAIDS) lower production and/orlevels of Aβ42 in cell cultures expressing Aβ40 and Aβ42 derived fromthe cleavage of APP. Since there is good evidence that high Aβ42 levelsare a major risk factor for AD, such drugs may be useful in preventing,delaying or reversing the progression of AD. The drawback of the use ofsuch drugs, however, is that large doses of NSAIDS are required forsignificant lowering of Aβ42, and significant gastrointestinal sideeffects, including bleeding ulcers, are associated with prolonged use ofNSAIDS at high doses (Langman et al., 1994, Lancet 343: 1075-1078). Inaddition, there remains an unknown risk for Alzheimer's disease due toamyloid formation from Aβ40 and other forms unaffected by Aβ42 loweringagents.

There is, therefore, a need in the art to develop treatments fordiseases or disorders related to the regulation of Aβ production.Furthermore, there is a need to develop methods of screening that can beused to identify compounds to treat such diseases or disorders.

Citation or identification of any reference in Section 2, or in anyother section of this application, shall not be considered an admissionthat such reference is available as prior art to the present invention.

3. SUMMARY OF THE INVENTION

The present invention is based, in part, on Applicants' surprisingdiscoveries that γ-secretase activity in cells requires adenosinetriphosphate (ATP) in order to optimally produce amyloid-β peptide(“Aβ”) from APP and that selective competitors of ATP reduce Aβ levels,in particular, levels of secreted Aβ.

The present invention, therefore, relates, generally, to methods andcompositions for preventing or treating an Aβ-related disorder such asAlzheimer's disease via administration of compounds that modulate, e.g.,inhibit, ATP-dependent enzymatic activity such as γ-secretase activity.

The invention provides a method for modulating amyloid-β peptide (Aβ)levels exhibited by a cell or tissue comprising contacting said cell ortissue with an amount of a compound sufficient to modulate said Aβlevels, wherein said compound modulates an ATP-dependent enzymaticactivity.

In one embodiment, the Aβ levels are lowered. In another embodiment, theAβ levels are raised. In another embodiment, the Aβ is Aβ40. In anotherembodiment, the Aβ is Aβ42.

In another embodiment, the modulating results in an increase in theratio of Aβ40 to Aβ42. In another embodiment, the modulating results inan increase in C99.

In another embodiment, the compound binds an enzyme that exhibits anATP-dependent enzymatic activity. In another embodiment, the compoundbinds a molecule that regulates the ATP-enzymatic activity.

In another embodiment, the compounds binds a molecule present in acomplex with an enzyme that exhibits the ATP-dependent enzymaticactivity, e.g., γ-secretase activity.

In another embodiment, the molecule is a molecule that is allostericallyregulated by ATP.

In another embodiment, the compound is an ATP modulator. In anotherembodiment, the the ATP modulator is a selective modulator.

In another embodiment, the ATP modulator competes with ATP for bindingto an ATP-binding site. In another embodiment, the ATP-binding site ispresent on an enzyme that exhibits an ATP-dependent enzymatic activity.In another embodiment, the ATP-binding site is present on a moleculethat regulates the ATP-dependent enzymatic activity.

In another embodiment, the compound does not affect total cellularlevels of β-amyloid precursor product (APP). In another embodiment, thecompound does not decrease levels of secreted APP (sAPP). In anotherembodiment, the compound increases levels of sAPPα. In anotherembodiment, levels of secreted Aβ are modulated. In another embodiment,the compound does not inhibit Notch-1 cleavage. In another embodiment,the compound does not inhibit tau phosphorylation.

In another embodiment, the compound crosses the blood-brain barrier.

In another embodiment, the enzymatic activity is a γ-secretase activity.In another embodiment, the compound inhibits γ-secretase activity. Inanother embodiment, the compound is an ATP modulator. In anotherembodiment, the ATP modulator is a selective modulator.

In another embodiment, the compound binds a γ-secretase enzymeexhibiting an ATP-dependent enzymatic activity. In another embodiment,the ATP modulator competes with ATP for binding to an ATP binding site.

In another embodiment, the compound is STI-571 or a blood-brain barrierpermeable variant thereof, or a pharmaceutically acceptable saltthereof. In another embodiment, the compound is a mesylate salt ofSTI-571. In another embodiment, the compound is WGB-BC-15 or ablood-brain barrier permeable variant thereof, or a pharmaceuticallyacceptable salt thereof. In another embodiment, the compound is Compound1 or a blood-brain barrier permeable variant thereof, or apharmaceutically acceptable salt thereof. In another embodiment, thecompound is Compound 2 or a blood-brain barrier permeable variantthereof, or a pharmaceutically acceptable salt thereof. In anotherembodiment, the compound is Compound 3 or a blood-brain barrierpermeable variant thereof, or a pharmaceutically acceptable saltthereof. In another embodiment, the compound is Compound 4 or ablood-brain barrier permeable variant thereof, or a pharmaceuticallyacceptable salt thereof. In another embodiment, the compound is Compound5 or a blood-brain barrier permeable variant thereof, or apharmaceutically acceptable salt thereof.

In another embodiment, the enzymatic activity is a kinase activity. Inanother embodiment, the compound binds to a kinase enzyme exhibiting anATP-dependent enzymatic activity. In another embodiment, the compoundcompetes with ATP for binding to an ATP-binding site on the kinase. Inanother embodiment, the compound binds to a site other than anATP-binding site on the kinase. In another embodiment, the kinase is atyrosine kinase. In another embodiment, the tyrosine kinase is Ablkinase, BCR-Abl kinase, ARG kinase, src kinase, c-kit orplatelet-derived growth factor receptor. In another embodiment, thekinase is a serine/threonine kinase, a carbohydrate kinase or a lipidkinase.

In another aspect, the invention provides a method for modulating Aβlevels exhibited by a cell or tissue comprising contacting said cell ortissue with an amount of an ATP modulator sufficient to modulate said Aβlevels.

In one embodiment, the Aβ levels are lowered. In another embodiment, theAβ levels are raised. In another embodiment, the Aβ is Aβ40. In anotherembodiment, the Aβ is Aβ42.

In another embodiment, the modulating results in an increase in theratio of Aβ40 to Aβ42. In another embodiment, the modulating results inan increase in C99. In another embodiment, the ATP modulator competeswith ATP for binding to an ATP-binding site.

In another embodiment, the ATP-binding site is present on an enzyme thatexhibits an ATP-dependent enzymatic activity. In another embodiment, theATP-binding site is present on a molecule that regulates theATP-dependent enzymatic activity. In another embodiment, the compounddoes not affect total cellular levels of β-amyloid precursor product(APP). In another embodiment, the compound does not decrease levels ofsecreted APP (sAPP). In another embodiment, the compound increaseslevels of sAPPα.

In another embodiment, levels of secreted Aβ are modulated.

In another embodiment, the compound does not inhibit Notch-1 cleavage.In another embodiment, the compound does not inhibit tauphosphorylation. In another embodiment, the compound crosses theblood-brain barrier. In another embodiment, the enzymatic activity is aγ-secretase activity. In another embodiment, the compound inhibitsγ-secretase activity.

In another embodiment, the the compound is an ATP modulator. In anotherembodiment, the ATP modulator is a selective modulator. In anotherembodiment, the compound binds a γ-secretase enzyme exhibiting anATP-dependent enzymatic activity.

In another embodiment, the compound is STI-571 or a blood-brain barrierpermeable variant thereof, or a pharmaceutically acceptable saltthereof. In another embodiment, the compound is a mesylate salt ofSTI-571. In another embodiment, the compound is WGB-BC-15 or ablood-brain barrier permeable variant thereof, or a pharmaceuticallyacceptable salt thereof. In another embodiment, the compound is any ofCompounds 1-5 or a blood-brain barrier permeable variant thereof, or apharmaceutically acceptable salt thereof.

In another embodiment, the enzymatic activity is a kinase activity. Inanother embodiment, the compound binds to a kinase enzyme exhibiting anATP-dependent enzymatic activity. In another embodiment, the compoundcompetes with ATP for binding to an ATP-binding site on the kinase. Inanother embodiment, the compound binds to a site other than anATP-binding site on the kinase. In another embodiment, the kinase is atyrosine kinase. In another embodiment, the tyrosine kinase is Ablkinase, BCR-Abl kinase, ARG kinase, src kinase, c-kit orplatelet-derived growth factor receptor. In another embodiment, thekinase is a serine/threonine kinase, a carbohydrate kinase or a lipidkinase. In another embodiment, the kinase is a serine/threonine kinase,a carbohydrate kinase or a lipid kinase.

In another embodiment, the compound competes with ATP for binding to atarget other than γ-secretase in the metabolic pathway that regulatesthe cleavage of APP to Aβ by γ-secretase. In specific embodiments, thetarget is a kinase (or kinase domain), a protease (e.g., an AAA proteaseof the kind localized to the inner mitochondrial membrane), aphosphatase, or a molecular chaperone molecule (e.g., hsp 60, hsp70,hsp90).

In one aspect in the invention provides a method for modulating Aβlevels in a cell or tissue comprising contacting said cell or tissuewith an amount of a compound sufficient to modulate said Aβ levels,wherein said compound contains the following pharmacophore:

-   -   wherein X is CH—, O, NH or N—CO—.

In one embodiment, said pharmacophore containing compound comprises acompound of Figure I or a pharmaceutically acceptable salt thereof:

Figure I

-   -   A is CH or N;    -   B and C are independently CH, N or N⁺—O—;    -   R¹ is H, SO₂R^(a), (C═O)_(r)O_(s)R^(a);    -   R², R³, R⁴ and R⁵ are independently H, OH, CHO, CN, halogen,        (C═O)_(r)O_(s)(C₁-C₁₀)alkyl, (C═O)_(r)O_(s)(C₂-C₁₀)alkenyl,        (C═O)_(r)O_(s)(C₂-C₁₀)alkynyl, (C═O)_(r)O_(s)cycloalkyl,        (C═O)_(r)O_(s)cycloalkenyl, (C═O)_(r)O_(s)cycloalkynyl,        (C═O)_(r)O_(s)heterocycyl, (C═O)_(r)O_(s)aryl,        (C═O)_(r)O_(s)heteroaryl, (C═O)_(r)O_(s)perfluoroalkyl or        (C₀-C₆)alkyl-NR^(b)R^(c),        wherein said alkykl, alkenyl, alkynyl, cycyloalkyl,        cycloalkenyl, cycloalkynyl, heterocycyl, aryl, heteroaryl and        perfluoroalkyl is optionally substituted with one or more        substituents selected from R⁶;    -   R⁶ is (C═O)_(r)O_(s)NR^(a)R^(b), (C═O)_(r)O_(s)aryl,        (C═O)_(r)O_(s)heterocycyl, halogen, OH, oxo,        (C═O)_(r)O_(s)(C₁-C₃)perfluoroalkyl, (C═O)_(r)O_(s)(C₁-C₆)alkyl,        CHO, CO₂H, CN, (C₀-C₆)alkyl-NR^(b)R^(c) or        (C₁-C₆)alkyl-heterocycyl,        wherein said alkyl-heterocycyl is optionally substituted with        OH;    -   R^(a) is (C₁-C₆)alkyl, aryl or heterocycyl; and    -   R^(b) and R^(c) independently are H,        (C═O)_(r)O_(s)(C₁-C₁₀)alkyl, SO₂R^(a),        (C═O)_(r)O_(s)heterocycyl, (C═O)_(r)O_(s)aryl,        (C═O)_(r)O_(s)heteroaryl or CO₂R^(a),        wherein r and s independently are 0 or 1 and said alkyl,        heterocycyl, aryl or heteroaryl is optionally substituted with        one or more substituents selected from R⁶.

In another embodiment, A is CH, B is N and C is CH.

In another embodiment, R¹ is H.

In another embodiment, R² is H and R³ is heteroaryl.

In another embodiment, R⁴ is (C═O)_(r)O_(s)(C₁-C₁₀)alkyl and R⁵ is(C₀-C₆)alkyl-NR^(b)R^(c).

In another embodiment, R³ is 3-pyridinyl.

In another embodiment, r is 0, s is 0 and (C₁-C₁₀)alkyl is methyl.

In another embodiment, (C₀-C₆)alkyl is a direct bond (C₀), R^(b) is Hand R^(c) is (C═O)_(r)O_(s)heteroaryl or (C═O)_(r)O_(s)heterocycyl.

In another embodiment, (C═O)_(r)O_(s)heterocycyl is4-hydroxy-1-piperazino, as illustrated below or a pharmaceuticallyacceptable salt thereof

In another embodiment, (C═O)_(r)O_(s)heteroaryl is 3-pyrindinyl, asillustrated below or a pharmaceutically acceptable salt thereof.

In another embodiment, said pharmacophore containing compound, comprisesa compound of Figure II or a pharmaceutically acceptable salt thereof:

Figure II

-   -   A, B and C are independently CH, N or N⁺—O—;    -   D is O, S or N—R⁵;    -   R¹ is H, SO₂R^(a), (C═O)_(r)R^(a) or CO₂R^(a);    -   R², R³ and R⁴ are independently H, OH, CHO, CN, halogen,        (C═O)_(r)O_(s)(C₁-C₁₀)alkyl, (C═O)_(r)O_(s)(C₂-C₁₀)alkenyl,        (C═O)_(r)O_(s)(C₂-C₁₀)alkynyl, (C═O)_(r)O_(s)cycloalkyl,        (C═O)_(r)O_(s)cycloalkenyl, (C═O)_(r)O_(s)cycloalkynyl,        (C═O)_(r)O_(s)heterocycyl, (C═O)_(r)O_(s)aryl,        (C═O)_(r)O_(s)heteroaryl, (C═O)_(r)O_(s)perfluoroalkyl or        (C₀-C₆)alkyl-NR^(b)R^(c),        wherein said alkykl, alkenyl, alkynyl, cycyloalkyl,        cycloalkenyl, cycloalkynyl, heterocycyl, aryl, heteroaryl and        perfluoroalkyl is optionally substituted with one or more        substituents selected from R⁶;    -   R⁵ is H, aryl or (C₁-C₆)alkyl;    -   R⁶ is (C═O)_(r)O_(s)NR^(a)R^(b), (C═O)_(r)O_(s)aryl,        (C═O)_(r)O_(s)heterocycyl, halogen, OH, oxo,        (C═O)_(r)O_(s)(C₁-C₃)perfluoroalkyl, (C═O)_(r)O_(s)(C₁-C₆)alkyl,        CHO, CO₂H, CN, (C₀-C₆)alkyl-NR^(b)R^(c) or        (C₁-C₆)alkyl-heterocycyl;    -   R^(a) is (C₁-C₆)alkyl, aryl or heterocycyl; and    -   R^(b) and R^(c) independently are H,        (C═O)_(r)O_(s)(C₁-C₁₀)alkyl, SO₂R^(a),        (C═O)_(r)O_(s)heterocycyl, (C═O)_(r)O_(s)aryl,        (C═O)_(r)O_(s)heteroaryl or CO₂R^(a),        wherein r and s independently are 0 or 1 and said alkyl,        heterocycyl, aryl or heteroaryl is optionally substituted with        one or more substituents selected from R⁶.

In another embodiment, R¹ is H.

In another embodiment, R² is H and R³ is (C₀-C₆)alkyl-NR^(b)R^(c).

In another embodiment, R⁴ is heteroaryl.

In another embodiment, said pharmacophore containing compound, comprisesa compound of Figure III or a pharmaceutically acceptable salt thereof:

Figure III

-   -   A is aryl or heteroaryl,        wherein said aryl or heteroaryl is optionally substituted with        one or more substituents selected from R³;    -   X is NH, N-acyl, O or S;    -   R¹ and R² are independently H, OH, CHO, CN, halogen,        (C═O)_(r)O_(s)(C₁-C₁₀)alkyl, (C═O)_(r)O_(s)(C₂-C₁₀)alkenyl,        (C═O)_(r)O_(s)(C₂-C₁₀)alkynyl, (C═O)_(r)O_(s)cycloalkyl,        (C═O)_(r)O_(s)cycloalkenyl, (C═O)_(r)O_(s)cycloalkynyl,        (C═O)_(r)O_(s)heterocycyl, (C═O)_(r)O_(s)aryl,        (C═O)_(r)O_(s)heteroaryl, (C═O)_(r)O_(s)perfluoroalkyl or        (C₀-C₆)alkyl-NR^(a)R^(b),        wherein said alkykl, alkenyl, alkynyl, cycyloalkyl,        cycloalkenyl, cycloalkynyl, heterocycyl, aryl, heteroaryl and        perfluoroalkyl is optionally substituted with one or more        substituents selected from R³;    -   R³ is (C═O)_(r)O_(s)NR^(a)R^(b), (C═O)_(r)O_(s)aryl,        (C═O)_(r)O_(s)heterocycyl, halogen, OH, oxo,        (C═O)_(r)O_(s)(C₁-C₃)perfluoroalkyl, (C═O)_(r)O_(s)(C₁-C₆)alkyl,        CHO, CO₂H, CN, (C₀-C₆)alkyl-NR^(a)R^(b) or        (C₁-C₆)alkyl-heterocycyl;    -   R^(a) and R^(b) independently are H,        (C═O)_(r)O_(s)(C₁-C₁₀)alkyl, SO₂R¹, (C═O)_(r)O_(s)heterocycyl,        (C═O)_(r)O_(s)aryl, (C═O)_(r)O_(s)heteroaryl or CO₂R¹,        wherein r, s and t independently are 0 or 1 and said alkyl,        heterocycyl, aryl or heteroaryl is optionally substituted with        one or more substituents selected from R³.

In another embodiment, A is aryl.

In another embodiment, the aryl is 2,5-dichlorophenyl.

In another embodiment, R¹ is (C₀-C₆)alkyl-NR^(a)R^(b) and R² is(C═O)_(r)O_(s)(C₁-C₁₀)alkyl.

In another embodiment, (C═O)_(r)O_(s)(C₁-C₁₀)alkyl is methyl.

In another embodiment, (C₀-C₆)alkyl-NR^(a)R^(b) is

or a pharmaceutically acceptable salt thereof.

In another aspect, the invention provides a pharmaceutical compositionuseful for modulating Aβ level in a cell or tissue, comprising:

-   -   an amount of a compound described in any one of claims 1 to 21        sufficient to modulate said Aβ level; and    -   a pharmaceutically acceptable excipient or carrier.

In one embodiment, the pharmaceutical composition is useful formodulating Aβ levels exhibited by a cell or tissue.

In another embodiment, the compound is present in an amount sufficientto modulate said Aβ levels.

Compounds according to Figure I may be prepared by various processesknown to one of skill in the art. In particular, compounds according toFigure I, wherein A is CH, B is N, C is CH and R¹ is H may be preparedby the following process:

-   -   a) a compound according to Figure IV        Figure IV

wherein Y and Z constitute (C₁-C₆)alkyl groups and R² and R³ are asabove-defined is reacted with a compound according to Figure VFigure V

wherein R⁴ and R⁵ are as above-defined.

In one embodiment, a compound utilized by the methods of the inventioncontains the following pharmacophore:

-   -   wherein X is CH—, O, NH or N—CO—.

In one embodiment, said pharmacophore containing compound comprises acompound of Figure I or a pharmaceutically acceptable salt thereof:Figure I

-   -   A is CH or N;    -   B and C are independently CH, N or N⁺—O—;    -   R¹ is H, SO₂R^(a), (C═O)_(r)O_(s)R^(a);    -   R², R³, R⁴ and R⁵ are independently H, OH, CHO, CN, halogen,        (C═O)_(r)O_(s)(C₁-C₁₀)alkyl, (C═O)_(r)O_(s)(C₂-C₁₀)alkenyl,        (C═O)_(r)O_(s)(C₂-C₁₀)alkynyl, (C═O)_(r)O_(s)cycloalkyl,        (C═O)_(r)O_(s)cycloalkenyl, (C═O)_(r)O_(s)cycloalkynyl,        (C═O)_(r)O_(s)heterocycyl, (C═O)_(r)O_(s)aryl,        (C═O)_(r)O_(s)heteroaryl, (C═O)_(r)O_(s)perfluoroalkyl or        (C₀-C₆)alkyl-NR^(b)R^(c),        wherein said alkykl, alkenyl, alkynyl, cycyloalkyl,        cycloalkenyl, cycloalkynyl, heterocycyl, aryl, heteroaryl and        perfluoroalkyl is optionally substituted with one or more        substituents selected from R⁶;    -   R⁶ is (C═O)_(r)O_(s)NR^(a)R^(b), (C═O)_(r)O_(s)aryl,        (C═O)_(r)O_(s)heterocycyl, halogen, OH, oxo,        (C═O)_(r)O_(s)(C₁-C₃)perfluoroalkyl, (C═O)_(r)O_(s)(C₁-C₆)alkyl,        CHO, CO₂H, CN, (C₀-C₆)alkyl-NR^(b)R^(c) or        (C₁-C₆)alkyl-heterocycyl,        wherein said alkyl-heterocycyl is optionally substituted with        OH;    -   R^(a) is (C₁-C₆)alkyl, aryl or heterocycyl; and    -   R^(b) and R^(c) independently are H,        (C═O)_(r)O_(s)(C₁-C₁₀)alkyl, SO₂R^(a),        (C═O)_(r)O_(s)heterocycyl, (C═O)_(r)O_(s)aryl,        (C═O)_(r)O_(s)heteroaryl or CO₂R^(a),        wherein r and s independently are 0 or 1 and said alkyl,        heterocycyl, aryl or heteroaryl is optionally substituted with        one or more substituents selected from R⁶.

In another embodiment, A is CH, B is N and C is CH.

In another embodiment, R¹ is H.

In another embodiment, R² is H and R³ is heteroaryl.

In another embodiment, R⁴ is (C═O)_(r)O_(s)(C₁-C₁₀)alkyl and R⁵ is(C₀-C₆)alkyl-NR^(b)R^(c).

In another embodiment, R³ is 3-pyridinyl.

In another embodiment, r is 0, s is 0 and (C₁-C₁₀)alkyl is methyl.

In another embodiment, (C₀-C₆)alkyl is a direct bond (C₀), R^(b) is Hand R^(c) is (C═O)_(r)O_(s)heteroaryl or (C═O)_(r)O_(s)heterocycyl.

In another embodiment, (C═O)_(r)O_(s)heterocycyl is4-hydroxy-1-piperazino, as illustrated below or a pharmaceuticallyacceptable salt thereof.

In another embodiment, (C═O)_(r)O_(s)heteroaryl is 3-pyrindinyl, asillustrated below or a pharmaceutically acceptable salt thereof.

In another embodiment, said pharmacophore containing compound, comprisesa compound of Figure II or a pharmaceutically acceptable salt thereof:

Figure II

-   -   A, B and C are independently CH, N or N⁺—O—;    -   D is O, S or N—R⁵;    -   R¹ is H, SO₂R^(a), (C═O)_(r)R^(a) or CO₂R^(a);    -   R², R³ and R⁴ are independently H, OH, CHO, CN, halogen,        (C═O)_(r)O_(s)(C₁-C₁₀)alkyl, (C═O)_(r)O_(s)(C₂-C₁₀)alkenyl,        (C═O)_(r)O_(s)(C₂-C₁₀)alkynyl, (C═O)_(r)O_(s)cycloalkyl,        (C═O)_(r)O_(s)cycloalkenyl, (C═O)_(r)O_(s)cycloalkynyl,        (C═O)_(r)O_(s)heterocycyl, (C═O)_(r)O_(s)aryl,        (C═O)_(r)O_(s)heteroaryl, (C═O)_(r)O_(s)perfluoroalkyl or        (C₀-C₆)alkyl-NR^(b)R^(c),        wherein said alkykl, alkenyl, alkynyl, cycyloalkyl,        cycloalkenyl, cycloalkynyl, heterocycyl, aryl, heteroaryl and        perfluoroalkyl is optionally substituted with one or more        substituents selected from R⁶;    -   R⁵ is H, aryl or (C₁-C₆)alkyl;    -   R⁶ is (C═O)_(r)O_(s)NR^(a)R^(b), (C═O)_(r)O_(s)aryl,        (C═O)_(r)O_(s)heterocycyl, halogen, OH, oxo,        (C═O)_(r)O_(s)(C₁-C₃)perfluoroalkyl, (C═O)_(r)O_(s)(C₁-C₆)alkyl,        CHO, CO₂H, CN, (C₀-C₆)alkyl-NR^(b)R^(c) or        (C₁-C₆)alkyl-heterocycyl;    -   R^(a) is (C₁-C₆)alkyl, aryl or heterocycyl; and    -   R^(b) and R^(c) independently are H,        (C═O)_(r)O_(s)(C₁-C₁₀)alkyl, SO₂R^(a),        (C═O)_(r)O_(s)heterocycyl, (C═O)_(r)O_(s)aryl,        (C═O)_(r)O_(s)heteroaryl or CO₂R^(a)        wherein r and s independently are 0 or 1 and said alkyl,        heterocycyl, aryl or heteroaryl is optionally substituted with        one or more substituents selected from R⁶.

In another embodiment, R¹ is H.

In another embodiment, R² is H and R³ is (C₀-C₆)alkyl-NR^(b)R^(c).

In another embodiment, R⁴ is heteroaryl.

In another embodiment, said pharmacophore containing compound, comprisesa compound of Figure III or a pharmaceutically acceptable salt thereof:

Figure III

-   -   A is aryl or heteroaryl,        wherein said aryl or heteroaryl is optionally substituted with        one or more substituents selected from R³;    -   X is NH, N-acyl, O or S;    -   R¹ and R² are independently H, OH, CHO, CN, halogen,        (C═O)_(r)O_(s)(C₁-C₁₀)alkyl, (C═O)_(r)O_(s)(C₂-C₁₀)alkenyl,        (C═O)_(r)O_(s)(C₂-C₁₀)alkynyl, (C═O)_(r)O_(s)cycloalkyl,        (C═O)_(r)O_(s)cycloalkenyl, (C═O)_(r)O_(s)cycloalkynyl,        (C═O)_(r)O_(s)heterocycyl, (C═O)_(r)O_(s)aryl,        (C═O)_(r)O_(s)heteroaryl, (C═O)_(r)O_(s)perfluoroalkyl or        (C₀-C₆)alkyl-NR^(a)R^(b),        wherein said alkykl, alkenyl, alkynyl, cycyloalkyl,        cycloalkenyl, cycloalkynyl, heterocycyl, aryl, heteroaryl and        perfluoroalkyl is optionally substituted with one or more        substituents selected from R³;    -   R³ is (C═O)_(r)O_(s)NR^(a)R^(b), (C═O)_(r)O_(s)aryl,        (C═O)_(r)O_(s)heterocycyl, halogen, OH, oxo,        (C═O)_(r)O_(s)(C₁-C₃)perfluoroalkyl,        (C═O)_(r)O_(s)S_(t)(C₁-C₆)alkyl, CHO, CO₂H, CN,        (C₀-C₆)alkyl-NR^(a)R^(b) or (C₁-C₆)alkyl-heterocycyl;    -   R^(a) and R^(b) independently are H,        (C═O)_(r)O_(s)(C₁-C₁₀)alkyl, SO₂R¹, (C═O)_(r)O_(s)heterocycyl,        (C═O)_(r)O_(s)aryl, (C═O)_(r)O_(s)heteroaryl or CO₂R¹,        wherein r, s and t independently are 0 or 1 and said alkyl,        heterocycyl, aryl or heteroaryl is optionally substituted with        one or more substituents selected from R³.

In another embodiment, A is aryl.

In another embodiment, the aryl is 2,5-dichlorophenyl.

In another embodiment, R¹ is (C₀-C₆)alkyl-NR^(a)R¹ and R² is(C═O)_(r)O_(s)(C₁-C₁₀)alkyl.

In another embodiment, (C═O)_(r)O_(s)(C₁-C₁₀)alkyl is methyl.

In another embodiment, (C₀-C₆)alkyl-NR^(a)R^(b) is

or a pharmaceutically acceptable salt thereof.

The invention also provides an article of manufacture comprisingpackaging material and a pharmaceutical composition comprising acompound that lowers ATP-dependent Aβ levels, and a pharmaceuticallyacceptable carrier contained within the packaging material, saidpharmaceutical composition in a form suitable for administration to asubject. In one embodiment, the article of manufacture further comprisesprinted instructions regarding the use or administration of thepharmaceutical composition. In one embodiment, the instructions suggesta dosing regimen for the prevention, treatment, or amelioration of asymptom of an Aβ-related disorder. In another embodiment, theinstructions suggest a dosing regimen for the prevention, treatment, oramelioration of a symptom of Alzheimer's disease.

In another embodiment, the article of manufacture further comprises alabel regarding the use or administration of the pharmaceuticalcomposition. In another embodiment, the label suggests a dosing regimenfor the prevention, treatment, or amelioration of a symptom of anAβ-related disorder. In another embodiment, the label suggests a dosingregimen for the prevention, treatment, or amelioration of a symptom ofAlzheimer's disease.

In another embodiment, the article of manufacture further comprises anantioxidant, a non-selective COX inhibitor or an acetylcholinesteraseinhibitor. In another embodiment, the pharmaceutical composition furthercomprises an antioxidant, a non-selective COX inhibitor or anacetylcholinesterase inhibitor.

The invention also provides method of identifying a compound thatmodulates Aβ levels exhibited by a cell or tissue comprising:

-   -   (a) determining a first level of γ-secretase activity in said        cell or tissue;    -   (b) contacting said cell or tissue with a test compound; and    -   (c) determining a second level of γ-secretase activity in said        cell or tissue,        wherein a difference in said first level and said second level        of γ-secretase activity is indicative of the ability of said        test compound to modulate Aβ levels.

In one embodiment, the difference in γ-secretase activity is indicativeof the ability of said test compound to modulate the Aβ levels. Inanother embodiment, Aβ levels are modulated. In another embodiment, theγ-secretase activity is an ATP-dependent enzymatic activity. In anotherembodiment, the γ-secretase activity is production of Aβ.

The invention also provides a method of identifying a compound thatmodulates Aβ levels exhibited by a cell or tissue comprising:

-   -   (a) contacting said cell or tissue with a test compound; and    -   (b) determining a level of activity of γ-secretase in said cell        or tissue;        wherein a difference in said level and a control level of        γ-secretase activity in a comparable cell or tissue not        contacted with the test compound is indicative of the ability of        said test compound to modulate Aβ levels.

In one embodiment, the difference in γ-secretase activity is indicativeof the ability of said test compound to modulate the Aβ levels. Inanother embodiment, Aβ levels are modulated. In another embodiment, theγ-secretase activity is an ATP-dependent enzymatic activity. In anotherembodiment, the γ-secretase activity is production of Aβ.

The invention also provides a method for identifying an agent to betested for an ability to treat an Aβ-related disorder in a patient inneed of such treatment comprising:

-   -   (a) contacting in a cell or tissue γ-secretase with a potential        agent; and    -   (b) detecting the amount of γ-secretase activity        wherein the agent is identified if a decrease in γ-secretase        activity is detected in the presence of the potential agent and        wherein the agent modulates Aβ levels.

In one embodiment, the ability to treat the Aβ-related disorder istested. In another embodiment, the γ-secretase activity is anATP-dependent enzymatic activity. In another embodiment, the γ-secretaseactivity is production of Aβ.

In another aspect, the invention provides a method for identifying anagent to be tested for an ability to modulate Aβ levels exhibited by acell or tissue comprising:

-   -   (a) determining a first level of γ-secretase activity in said        cell or tissue;    -   (b) contacting said cell or tissue with a potential agent; and    -   (c) determining a second level of γ-secretase activity in said        cell or tissue, wherein a difference in said first level and        said second level of γ-secretase activity is indicative of the        ability of said potential agent to modulate Aβ levels. In one        embodiment, the method comprises the additional step of:    -   (d) determining whether the Aβ levels are modulated.

In another embodiment, the γ-secretase activity is an ATP-dependentenzymatic activity. In another embodiment, the γ-secretase activity isproduction of Aβ.

The invention also provides a method for identifying an agent to betested for an ability to modulate Aβ levels exhibited by a cell ortissue comprising:

-   -   (a) contacting said cell or tissue with a potential agent; and    -   (b) determining a level of γ-secretase activity in said cell or        tissue;        wherein a difference in said level and a control level of        γ-secretase activity in a comparable cell or tissue not        contacted with the test compound is indicative of the ability of        said potential agent to modulate Aβ levels.

In one embodiment, the method comprises the additional step of:

-   -   (c) determining whether the Aβ levels are modulated.

In another embodiment, the γ-secretase activity is an ATP-dependentenzymatic activity.

In another embodiment, the γ-secretase activity is production of Aβ.

The invention also provides a method for selecting a potentialtherapeutic agent for use in the treatment of an Aβ-related disordercomprising:

-   -   (a) administering a potential therapeutic agent to an animal;    -   (b) measuring the response of said animal to said potential        therapeutic agent;    -   (c) comparing the response of said animal with that of a control        animal to which the potential therapeutic agent has not been        administered; and    -   (d) selecting a potential therapeutic agent based on the        difference in responses observed between said animal and said        control animal,        wherein the potential therapeutic agent modulates ATP-dependent        γ-secretase activity.

In one embodiment, the animal is a guinea pig. In another embodiment,the disorder is Alzheimer's disease.

The invention also provides a method for selecting a potentialtherapeutic agent for use in the treatment of an Aβ-related disordercomprising:

-   -   (a) administering a potential therapeutic agent to an animal;    -   (b) measuring the response of said animal to administration of        an agent that modulates activity of γ-secretase;    -   (c) comparing the response of said animal with that of a control        animal to which the potential therapeutic agent has not been        administered; and    -   (d) selecting a potential therapeutic agent based on the        difference in responses observed between said animal and said        control animal,        wherein the potential therapeutic agent modulates ATP-dependent        γ-secretase activity.

In one embodiment, the animal is a guinea pig. In another embodiment,the disorder is Alzheimer's disease.

The invention also provides a method for selecting a potentialtherapeutic agent for use in the treatment of an Aβ-related disordercomprising:

-   -   (a) administering a potential therapeutic agent to an animal;    -   (b) measuring the response of said animal, wherein the response        is selected from the group consisting of:        -   (i) exhibition of behavior in a Morris water maze; and        -   (i) exhibition of behavior in a Y-maze    -   (c) comparing the response of said animal with that of a control        animal to which the potential therapeutic agent has not been        administered; and    -   (d) selecting a potential therapeutic agent based on the        difference in responses observed between said animal and said        control animal,        wherein the potential therapeutic agent modulates Aβ levels.

In one embodiment, the animal is a guinea pig. In another embodiment,the disorder is Alzheimer's disease.

The invention also provides a method for preventing, delaying orreversing the progression of an Aβ-related disorder comprising:

-   -   (a) identifying a mammal in need of prevention, delay, or        reversal of the progression of the disorder; and    -   (b) administering to said mammal an amount of an agent        sufficient to modulate ATP-dependent γ-secretase activity,        wherein Aβ levels are modulated.

In one embodiment, the agent inhibits or decreases ATP-dependentγ-secretase activity.

In another embodiment, the mammal is human. In another embodiment, thedisorder is Alzheimer's disease.

In another embodiment, the agent promotes or increases ATP-dependentγ-secretase activity.

In another embodiment, the agent is administered orally.

In another embodiment, the agent is administered with a NSAID. Inspecific embodiment, the NSAID is sulindac sulfide, flufenamic acid,ibuprofen, flurbiprofen, fenoprofen, mefenamic acid, indomethacin,carprofen, meclofenamic acid.

In another embodiment, the agent is administered with an antioxidant. Inanother embodiment, the antioxidant is selected from the groupconsisting of vitamin E, vitamin C, curcumin, and Gingko biloba.

In another embodiment, the agent the agent is administered with anacetylcholinesterase inhibitor.

The invention also provides a method of treating, or ameliorating asymptom of, an Aβ-related disorder comprising administering to a subjectin need of such treating or ameliorating an amount of a compoundsufficient to reduce Aβ levels in the subject, wherein the compoundmodulates an ATP-dependent enzymatic activity, such that the Aβ-relateddisorder is treated or a symptom of the Aβ related disorder isameliorated.

In one embodiment, the Aβ related disorder is Alzheimer's disease. Inanother embodiment, progression of the Aβ related disorder is slowed. Inanother embodiment, progression of the Aβ related disorder is reversed.

In another embodiment, the subject is a human subject. In a specificembodiment, the subject is a subject at risk for a familial form ofAlzheimer's disease.

In another embodiment, the ATP-dependent enzymatic activity is lowered.In another embodiment, the compound crosses the blood-brain barrier. Inanother embodiment, the compound is administered orally.

In another embodiment, the compound is administered with a NSAID. In aspecific embodiment, the NSAID is sulindac sulfide, flufenamic acid,ibuprofen, flurbiprofen, fenoprofen, mefenamic acid, indomethacin,carprofen, meclofenamic acid.

In another embodiment, the compound is administered with an antioxidant.In a specific embodiment, the antioxidant is selected from the groupconsisting of vitamin E, vitamin C, curcumin, and Gingko biloba.

In another embodiment, the compound is administered with anacetylcholinesterase inhibitor.

In another embodiment, the compound is an ATP modulator. In anotherembodiment, the compound is ATP modulator is a selective modulator.

In another embodiment, the enzymatic activity is a γ-secretase activity.In another embodiment, the compound binds a γ-secretase enzymeexhibiting an ATP-dependent enzymatic activity.

In another embodiment, the ATP modulator competes with ATP for bindingto an ATP binding site.

In one embodiment, the compound is STI-571 or a blood-brain barrierpermeable variant thereof, or a pharmaceutically acceptable saltthereof. In another embodiment, the compound is a mesylate salt ofSTI-571. In another embodiment, the compound is WGB-BC-15 or ablood-brain barrier permeable variant thereof, or a pharmaceuticallyacceptable salt thereof. In another embodiment, the compound is any ofCompounds 1-5 or a blood-brain barrier permeable variant thereof, or apharmaceutically acceptable salt thereof.

In one embodiment, the enzymatic activity is a kinase activity. In oneembodiment, the compound binds to a kinase enzyme exhibiting anATP-dependent enzymatic activity. In one embodiment, the compoundcompetes with ATP for binding to an ATP-binding site on the kinase. Inanother embodiment, the compound binds to a site other than anATP-binding site on the kinase.

In a specific embodiment, the kinase is a tyrosine kinase. In anotherembodiment, the tyrosine kinase is Abl kinase, BCR-Abl kinase, ARGkinase, src kinase, c-kit, platelet-derived growth factor receptor.

In a specific embodiment, the kinase is a serine/threonine kinase, acarbohydrate kinase or a lipid kinase.

In another embodiment, the enzymatic activity is activity of a protease(e.g., AAA protease of the kind localized to the inner mitochondrialmembrane), phosphatase, or a molecular chaperone molecule (e.g., hsp 60,hsp70, hsp90).

The invention also provides a method of treating, or ameliorating asymptom of, an Aβ-related disorder comprising administering to a subjectin need of such treating or ameliorating an amount of an ATP modulatorsufficient to reduce Aβ levels in the subject, such that the Aβ-relateddisorder is treated or a symptom of the Aβ related disorder isameliorated.

In one embodiment, the Aβ related disorder is Alzheimer's disease. Inone embodiment, the subject is a human subject. In another embodiment,

In one embodiment, the subject is a subject at risk for a familial formof Alzheimer's disease.

In another embodiment, the ATP-dependent enzymatic activity is lowered.

In another embodiment, the ATP modulator crosses the blood-brainbarrier.

In another embodiment, the ATP modulator is administered orally. Inanother embodiment, the ATP modulator is administered with a NSAID. Inanother embodiment, the NSAID is sulindac sulfide, flufenamic acid,ibuprofen, flurbiprofen, fenoprofen, mefenamic acid, indomethacin,carprofen, meclofenamic acid.

In another embodiment, the ATP modulator is administered with anantioxidant. In another embodiment, the antioxidant is selected from thegroup consisting of vitamin E, vitamin C, curcumin, and Gingko biloba.

In another embodiment, the ATP modulator is administered with anacetylcholinesterase inhibitor.

In another embodiment, the ATP modulator is a selective modulator.

In another embodiment, the enzymatic activity is a γ-secretase activity.

In one embodiment, the compound binds a γ-secretase enzyme exhibiting anATP-dependent enzymatic activity. In another embodiment, the ATPmodulator competes with ATP for binding to an ATP binding site.

In one embodiment, the compound is STI-571 or a blood-brain barrierpermeable variant thereof, or a pharmaceutically acceptable saltthereof. In another embodiment, the compound is a mesylate salt ofSTI-571. In another embodiment, the compound is WGB-BC-15 or ablood-brain barrier permeable variant thereof, or a pharmaceuticallyacceptable salt thereof. In another embodiment, the compound is any ofCompounds 1-5 or a blood-brain barrier permeable variant thereof, or apharmaceutically acceptable salt thereof.

In one embodiment, the enzymatic activity is a kinase activity. In oneembodiment, the compound binds to a kinase enzyme exhibiting anATP-dependent enzymatic activity. In one embodiment, the compoundcompetes with ATP for binding to an ATP-binding site on the kinase. Inanother embodiment, the compound binds to a site other than anATP-binding site on the kinase.

In a specific embodiment, the kinase is a tyrosine kinase. In anotherembodiment, the tyrosine kinase is Abl kinase, BCR-Abl kinase, ARGkinase, src kinase, c-kit, platelet-derived growth factor receptor.

In a specific embodiment, the kinase is a serine/threonine kinase, acarbohydrate kinase or a lipid kinase.

In another embodiment, the enzymatic activity is activity of a protease(e.g., AAA protease of the kind localized to the inner mitochondrialmembrane), phosphatase, or a molecular chaperone molecule (e.g., hsp 60,hsp70, hsp90).

The invention also provides a method of treating, or ameliorating asymptom of, an Aβ-related disorder comprising administering to a subjectin need of such treating or ameliorating an amount of a compoundsufficient to reduce Aβ levels in the subject, such that the Aβ-relateddisorder is treated or a symptom of the Aβ-related disorder isameliorated, wherein the compound contains the following pharmacophore:

-   -   wherein X is CH—, O, NH or N—CO—.

In one embodiment, said pharmacophore containing compound comprises acompound of Figure I or a pharmaceutically acceptable salt thereof:

Figure I

-   -   A is CH or N;    -   B and C are independently CH, N or N⁺—O—;    -   R¹ is H, SO₂R^(a), (C═O)_(r)O_(s)R^(a);    -   R², R³, R⁴ and R⁵ are independently H, OH, CHO, CN, halogen,        (C═O)_(r)O_(s)(C₁-C₁₀)alkyl, (C═O)_(r)O_(s)(C₂-C₁₀)alkenyl,        (C═O)_(r)O_(s)(C₂-C₁₀)alkynyl, (C═O)_(r)O_(s)cycloalkyl,        (C═O)_(r)O_(s)cycloalkenyl, (C═O)_(r)O_(s)cycloalkynyl,        (C═O)_(r)O_(s)heterocycyl, (C═O)_(r)O_(s)aryl,        (C═O)_(r)O_(s)heteroaryl, (C═O)_(r)O_(s)perfluoroalkyl or        (C₀-C₆)alkyl-NR^(b)R^(c),        wherein said alkykl, alkenyl, alkynyl, cycyloalkyl,        cycloalkenyl, cycloalkynyl, heterocycyl, aryl, heteroaryl and        perfluoroalkyl is optionally substituted with one or more        substituents selected from R⁶;    -   R⁶ is (C═O)_(r)O_(s)NR^(a)R^(b), (C═O)_(r)O_(s)aryl,        (C═O)_(r)O_(s)heterocycyl, halogen, OH, oxo,        (C═O)_(r)O_(s)(C₁-C₃)perfluoroalkyl, (C═O)_(r)O_(s)(C₁-C₆)alkyl,        CHO, CO₂H, CN, (C₀-C₆)alkyl-NR^(b)R^(c) or        (C₁-C₆)alkyl-heterocycyl,        wherein said alkyl-heterocycyl is optionally substituted with        OH;    -   R^(a) is (C₁-C₆)alkyl, aryl or heterocycyl; and    -   R^(b) and R^(c) independently are H,        (C═O)_(r)O_(s)(C₁-C₁₀)alkyl, SO₂R^(a),        (C═O)_(r)O_(s)heterocycyl, (C═O)_(r)O_(s)aryl,        (C═O)_(r)O_(s)heteroaryl or CO₂R^(a),        wherein r and s independently are 0 or 1 and said alkyl,        heterocycyl, aryl or heteroaryl is optionally substituted with        one or more substituents selected from R⁶.

In another embodiment, A is CH, B is N and C is CH.

In another embodiment, R¹ is H.

In another embodiment, R² is H and R³ is heteroaryl.

In another embodiment, R⁴ is (C═O)_(r)O_(s)(C₁-C₁₀)alkyl and R⁵ is(CO—C₆)alkyl-NR^(b)R^(c).

In another embodiment, R³ is 3-pyridinyl.

In another embodiment, r is 0, s is 0 and (C₁-C₁₀)alkyl is methyl.

In another embodiment, (C₀-C₆)alkyl is a direct bond (C₀), R^(b) is Hand R^(c) is (C═O)_(r)O_(s)heteroaryl or (C═O)_(r)O_(s)heterocycyl.

In another embodiment, (C═O)_(r)O_(s)heterocycyl is4-hydroxy-1-piperazino, as illustrated below or a pharmaceuticallyacceptable salt thereof.

In another embodiment, the pharmacophore containing compound comprises acompound of Figure II or a pharmaceutically acceptable salt thereof:

Figure II

-   -   A, B and C are independently CH, N or N⁺—O—;    -   D is O, S or N—R⁵;    -   R¹ is H, SO₂R^(a), (C═O)R^(a) or CO₂R^(a);    -   R², R³ and R⁴ are independently H, OH, CHO, CN, halogen,        (C═O)_(r)O_(s)(C₁-C₁₀)alkyl, (C═O)_(r)O_(s)(C₂-C₁₀)alkenyl,        (C═O)_(r)O_(s)(C₂-C₁₀)alkynyl, (C═O)_(r)O_(s)cycloalkyl,        (C═O)_(r)O_(s)cycloalkenyl, (C═O)_(r)O_(s)cycloalkynyl,        (C═O)_(r)O_(s)heterocycyl, (C═O)_(r)O_(s)aryl,        (C═O)_(r)O_(s)heteroaryl, (C═O)_(r)O_(s)perfluoroalkyl or        (C₀-C₆)alkyl-NR^(b)R^(c),        wherein said alkykl, alkenyl, alkynyl, cycyloalkyl,        cycloalkenyl, cycloalkynyl, heterocycyl, aryl, heteroaryl and        perfluoroalkyl is optionally substituted with one or more        substituents selected from R⁶;    -   R⁵ is H, aryl or (C₁-C₆)alkyl;    -   R⁶ is (C═O)_(r)O_(s)NR^(a)R^(b), (C═O)_(r)O_(s)aryl,        (C═O)_(r)O_(s)heterocycyl, halogen, OH, oxo,        (C═O)_(r)O_(s)(C₁-C₃)perfluoroalkyl, (C═O)_(r)O_(s)(C₁-C₆)alkyl,        CHO, CO₂H, CN, (C₀-C₆)alkyl-NR^(b)R^(c) or        (C₁-C₆)alkyl-heterocycyl;    -   R^(a) is (C₁-C₆)alkyl, aryl or heterocycyl; and

R^(b) and R^(c) independently are H, (C═O)_(r)O_(s)(C₁-C₁₀)alkyl,SO₂R^(a), (C═O)_(r)O_(s)heterocycyl, (C═O)_(r)O_(s)aryl,(C═O)_(r)O_(s)heteroaryl or CO₂R^(a),

wherein r and s independently are 0 or 1 and said alkyl, heterocycyl,aryl or heteroaryl is optionally substituted with one or moresubstituents selected from R⁶.

In another embodiment, R¹ is H.

In another embodiment, R² is H and R³ is (C₀-C₆)alkyl-NR^(b)R^(c).

In another embodiment, R⁴ is heteroaryl.

In another embodiment, said pharmacophore containing compound, comprisesa compound of Figure III or a pharmaceutically acceptable salt thereof:

Figure III

-   -   A is aryl or heteroaryl,        wherein said aryl or heteroaryl is optionally substituted with        one or more substituents selected from R³;    -   X is NH, N-acyl, O or S;    -   R¹ and R² are independently H, OH, CHO, CN, halogen,        (C═O)_(r)O_(s)(C₁-C₁₀)alkyl, (C═O)_(r)O_(s)(C₂-C₁₀)alkenyl,        (C═O)_(r)O_(s)(C₂-C₁₀)alkynyl, (C═O)_(r)O_(s)cycloalkyl,        (C═O)_(r)O_(s)cycloalkenyl, (C═O)_(r)O_(s)cycloalkynyl,        (C═O)_(r)O_(s)heterocycyl, (C═O)_(r)O_(s)aryl,        (C═O)_(r)O_(s)heteroaryl, (C═O)_(r)O_(s)perfluoroalkyl or        (C₀-C₆)alkyl-NR^(a)R^(b),        wherein said alkykl, alkenyl, alkynyl, cycyloalkyl,        cycloalkenyl, cycloalkynyl, heterocycyl, aryl, heteroaryl and        perfluoroalkyl is optionally substituted with one or more        substituents selected from R³;    -   R³ is (C═O)_(r)O_(s)NR^(a)R^(b), (C═O)_(r)O_(s)aryl,        (C═O)_(r)O_(s)heterocycyl, halogen, OH, oxo,        (C═O)_(r)O_(s)(C₁-C₃)perfluoroalkyl,        (C═O)_(r)O_(s)S_(t)(C₁-C₆)alkyl, CHO, CO₂H, CN,        (C₀-C₆)alkyl-NR^(a)R^(b) or (C₁-C₆)alkyl-heterocycyl;    -   R^(a) and R^(b) independently are H,        (C═O)_(r)O_(s)(C₁-C₁₀)alkyl, SO₂R¹, (C═O)_(r)O_(s)heterocycyl,        (C═O)_(r)O_(s)aryl, (C═O)_(r)O_(s)heteroaryl or CO₂R¹,        wherein r, s and t independently are 0 or 1 and said alkyl,        heterocycyl, aryl or heteroaryl is optionally substituted with        one or more substituents selected from R³.

In another embodiment, A is aryl.

In another embodiment, aryl is 2,5-dichlorophenyl.

In another embodiment, R¹ is (C₀-C₆)alkyl-NR^(a)R^(b) and R² is(C═O)_(r)O_(s)(C₁-C₁₀)alkyl.

In another embodiment, (C═O)_(r)O_(s)(C₁-C₁₀)alkyl is methyl.

In another embodiment, (C₀-C₆)alkyl-NR^(a)R^(b) is

or a pharmaceutically acceptable salt thereof.

In another embodiment, the agent is administered orally.

In one embodiment, the compound is administered with a NSAID. In anotherembodiment, the NSAID is sulindac sulfide, flufenamic acid, ibuprofen,flurbiprofen, fenoprofen, mefenamic acid, indomethacin, carprofen,meclofenamic acid.

In one embodiment, the compound is administered with an antioxidant. Ina specific embodiment, the antioxidant is selected from the groupconsisting of vitamin E, vitamin C, curcumin, and Gingko biloba.

In another embodiment, the compound is administered with anacetylcholinesterase inhibitor.

3.1. Terminology

As used herein, enzymatic activity of an enzyme, e.g., γ-secretase, is“ATP-dependent” if (i) the activation state (level of activity) of theenzyme is modulated by the binding of ATP to the enzyme; (ii) theactivity of the enzyme is modulated by the binding of ATP to a moleculein an enzyme complex that contains the enzyme; (iii) the activity of theenzyme is modulated by the binding of ATP to a molecule that effects theassembly, localization or stability of the enzyme complex; or (iv) theactivity of the enzyme is modulated by the binding of ATP to a moleculethat affects the activation state (level of activity) of the enzymecomplex.

As used herein, the terms “modulate,” “modulates,” “modulated” or“modulation” shall have their usual meanings, and encompass the meaningsof the words “enhance,” “promote,” “increase,” “agonize,” “inhibit,”“decrease” or “antagonize.”

As used herein, an “agonist” is any compound that acts directly orindirectly on a molecule to produce a pharmacological effect, while an“antagonist” is any compound that blocks the stimulation of apharmacological effect.

As used herein, a “sufficient amount” of a compound, or “an amount of acompound sufficient to . . . ” refers to an amount that contains atleast the minimum amount necessary to achieve the intended result. Suchan amount can routinely be determined by one of skill in the art basedon data from studies using methods of analysis such as those disclosedherein. Such data may include, but not be limited to, results from IC50determinations, as discussed hereinbelow.

As used herein, the term “Aβ-related disorder” or an “Aβ disorder” is adisease (e.g., Alzheimer's disease) or a condition (e.g., seniledementia) that involves an aberration or dysregulation of Aβ levels. AnAβ-related disorder includes, but is not limited to Alzheimer's disease,Down's syndrome and inclusion body myositis.

As used herein, the terms “disorder” and “disease” are usedinterchangeably to refer to a condition in a subject.

As used herein, a “small organic molecule” is an organic compound (ororganic compound complexed with an inorganic compound (e.g., metal))that generally has a molecular weight of less than about 3 kilodaltons,preferably less than about 1.5 kilodaltons. Preferably, the smallorganic molecule can be orally administered and/or cross the blood-brainbarrier.

As used herein, the term “about” means within 10 to 15%, preferablywithin 5 to 10%.

As used herein, the term “ATP modulator,” refers to a compound thatmodulates ATP function in a cell. For example, an ATP modulator can be acompound that binds to a molecule normally affected by ATP binding and,by binding to the molecule, modulates, e.g., inhibits, ATP's usualeffect on the molecule. Such an ATP modulator can bind the molecule atan ATP-binding site on the molecule or at some other site on themolecule. The ATP modulator can act by competing with ATP for binding tothe molecule, e.g., by competing for binding to an ATP-binding site.Preferably the ATP modulator is a selective ATP modulator.

As used herein, the terms “selective ATP modulator” or “selective ATPmodulation” refers to a modulator (or modulation) that discriminatesamong the molecules that affect ATP function in the cell and onlymodulates a specific individual molecule, or class or subset of themolecules (a “modulatory profile”). For example, a compound may beassayed for activity in inhibiting a collection of various kinases, andis recognized as a selective ATP modulator if it modulates ATP functionin a related class of kinases. In one embodiment, a selective ATPmodulator is one that exhibits a modulatory profile/identical to orsubstantially similar to that exhibited by STI-571, a mesylate salt ofSTI-571, Compound 1 or Compound 2.

As used herein, the terms “treat”, “treatment” and “treating” refer tothe amelioration of a Aβ-related disorder that results from theadministration of one or more compounds that lower Aβ levels exhibitedby a cell or tissue. It also refers to the managing of the disorder, orthe slowing and/or reversing of the progression of the disorder.

Symptoms of Aβ-related disorders are well known to those of skill in theart. For example, symptoms of Alzheimer's disease are well known in theart and can include, e.g., memory loss, mild cognitive impairment,cognitive decline, severe cognitive impairment and personality changesthat result in loss of functional ability, e.g., over the course of adecade. In debilitated states, patients usually exhibit severeimpairment, and retain only vegetative neurologic function. Symptoms ofAlzheimer's disease can also include certain art-known neuropathologicallesions, including intracellular neurofibrillary tangles andextracellular parenchymal and cerebrovascular amyloid.

As used herein, the terms “manage,” “managing” and “management” refer tothe beneficial effects that a subject derives from a compound, such as acompound that lowers Aβ levels exhibited by a cell or tissue, which doesnot result in a cure of the disease. In certain embodiments, a subjectis administered one or more such agents to “manage” a disorder so as toprevent or slow the progression or worsening of the disorder.

As used herein, the terms “prevent”, “preventing” and “prevention” referto the impedition of the recurrence or onset of an Aβ-related disorderor one or more symptoms of a Aβ-related disorder in a subject.

A used herein, a “protocol” includes dosing schedules and dosingregimens. The protocols herein are methods of use and includeprophylactic and therapeutic protocols.

As used herein, the terms “subject” and “patient” are usedinterchangeably. As used herein, the terms “subject” and “subjects”refer to an animal, preferably a mammal including a non-primate (e.g., acow, pig, horse, donkey, goat, camel, cat, dog, guinea pig, rat, mouse,sheep) and a primate (e.g., a monkey, such as a cynomolgous monkey,gorilla, chimpanzee, and a human), preferably a human. In oneembodiment, the subject is a subject with Alzheimer's disease.

The term “pharmaceutically acceptable” as used herein means acomposition, e.g., a carrier, excipient, or salt, approved by aregulatory agency of the Federal or a state government or listed in theU.S. Pharmacopoeia or other generally recognized pharmacopoeia for usein animals, and more particularly, in humans.

4. DESCRIPTION OF THE FIGURES

FIG. 1 is a bar graph quantitating the effect of adding anATP-regenerating system to a cell-free assay system for Aβ generation.The system is derived from mouse N2a cells expressing both the Swedishvariant of human APP and the presenilin-1 (PS1) mutation.

FIG. 2 is an autoradiogram showing that the β-secretase cleavageproduct, C99 (also known as βCTF), accumulates in an ATP-depleted cellfree system.

FIG. 3 is a bar graph showing strong ATP-dependence of C99 cleavage toAβ in isolated membranes.

FIG. 4 is a graph of the dose-response for reduction of Aβ levels inisolated membranes by Compound 1.

FIG. 5 is a mass spectrographic profile of peptides immunoprecipitatedfrom the supernatant of N2a cells, expressing both the Swedish variantof human APP and the presenilin-1 (PS 1) mutation, with anti-Aβ antibody6E 10 (Senetek, Napa, Calif.), showing decreased Aβ40 (“Aβ 1-40”) andAβ42 (“Aβ1-42”) in the presence of Compound 1. Y-axis, concentration ofCompound 1 (M).

FIG. 6 is a graph of the dose-dependent inhibition by the mesylate saltof STI-571 (imatinib mesylate, GLEEVEC™, Novartis Pharmaceuticals;Calbiochem) of Aβ formation by intact N2a cells expressing the Swedishvariant of human APP. The graph also demonstrates that APP levels do notchange in response to STI-571 administration.

FIG. 7 is a graph of the dose-dependent inhibition by Compound 1 of Aβformation in intact N2a cells expressing the Swedish variant of humanAPP. The graph also demonstrates that APP levels do not change inresponse to STI-571 administration.

FIGS. 8A-B. FIG. 8A is a bar graph showing the reduction of Aβ producedfrom primary neuronal cells cultured from rat brain in response toCompound 1. FIG. 8B is an autoradiogram showing the amount of APPproduced (top panel) and the amount of Aβ produced at differentconcentrations of Compound 1 (bottom panel).

FIG. 9 is an autoradiogram illustrating consistent secretion of solubleAPP-α (“APPa”) from primary neuronal cells cultured from rat brain inthe presence of Compound 1.

FIG. 10 is a bar graph showing the reduction of Aβ produced from primaryneuronal cells cultured from rat brain in response to the presence ofCompound 2.

FIG. 11 is a bar graph showing the time course (in hours) of the effectof STI-571 (mesylate salt) on Aβ generation from primary neuronal cellscultured from rat brain.

FIG. 12 is a bar graph showing the time course of Compound 1 effect onAβ generation from primary neuronal cells cultured from rat brain.

FIG. 13 is a bar graph showing the dose response to STI-571 on Aβgeneration from primary neuronal cells cultured from rat brain.

FIG. 14 is a bar graph showing the dose response to Compound 1 on Aβgeneration from primary neuronal cells cultured from rat brain. X-axis,DMSO control and Compound 1 concentration (M).

FIGS. 15A-D show the quantitation of STI-571 mesylate salt(“STI”)-dependent decreases in Aβ1-40(15A), Aβ 1-42 (15B) and increasesin C99 (15C and 15D), in vivo in guinea pig. FIG. 15C also demonstratesthat full-length βAPP levels do not change in response to treatment. **,p<0.01; *, 0.01<p<0.05.

FIGS. 16A-D show the quantitation of Compound 1 (“CMPD 1”)-dependentdecreases in A1-40(16A), Aβ1-42 (16B) and increases in C99 (16C and16D), in vivo in guinea pig. FIG. 16C also demonstrates that full-lengthβAPP levels do not change in response to treatment.*, p<0.01; *,0.01<p<0.05.

FIG. 17 is a graph and an autoradiogram showing stable levels of Notchcleavage in intact cells treated with STI-571 (mesylate salt). Thefigure presents results demonstrating that while STI-571 administrationreduces Aβ levels in a dose-dependent manner, the level of Notch andNotch cleavage product (Notch intracellular domain, NICD) remainsstable.

FIG. 18 is a graph showing stable levels of Notch cleavage in intactcells treated with Compound 1.

FIG. 19 is an autoradiogram illustrating that a standard γ-secretaseinhibitor (“γ-secretase inhibitor”; Calbiochem) inhibits Notch cleavagein a cell-free system derived from mΔe-Notch 1 transfected N2a cells,and that γ-secretase activity is therefore necessary for Notch cleavagein this system. The transfected cells were permeabilized and incubatedeither with or without a standard ATP energy regenerating system withGTP, and either in the presence or absence of a standard γ-secretaseinhibitor (“γ-secretase inhibitor,” Calbiochem). γ-sec, γ-secretase.ERS, energy regenerating system. NICD, Notch intracellular domain.

FIG. 20 is a diagram of the chemical structures of ST-571 (known in itsmesylate salt form as the active ingredient in GLEEVECT™ capsules),STI-571 variant (“WGB-BC-15”), Compound 1 (PD173955, Moasser et al.,1999, Cancer Research 59: 6145-6152; Wisniewski et al., 2002, CancerResearch 62(15):4244-55), and Compound 2 (PD166326; Wisniewski et al.,2002, Cancer Research 62(15):4244-55).

5. DETAILED DESCRIPTION OF THE INVENTION

For clarity of disclosure, and not by way of limitation, the detaileddescription of the invention is divided into the subsections set forthbelow.

5.1. Methods for Modulating Amyloid β Peptide (Aβ) Levels

The invention provides a method for modulating amyloid-β peptide (Aβ)levels exhibited by a cell or tissue comprising contacting said cell ortissue with an amount of a compound sufficient to modulate said Aβlevels, wherein said compound modulates an ATP-dependent enzymaticactivity.

A cell or tissue may include, but not be limited to: an excitable cell,e.g., a sensory neuron, motomeuron, or interneuron; a glial cell; aprimary culture of cells, e.g., a primary culture of neuronal or glialcells; cell(s) derived from a neuronal or glial cell line; dissociatedcell(s); whole cell(s) or intact cell(s); permeabilized cell(s); abroken cell preparation; an isolated and/or purified cell preparation; acellular extract or purified enzyme preparation; a tissue or organ,e.g., brain, brain structure, brain slice, spinal cord, spinal cordslice, central nervous system, peripheral nervous system, or nerve;tissue slices, and a whole animal. In certain embodiments, the brainstructure is cerebral cortex, the hippocampus, or their anatomicaland/or functional counterparts in other mammalian species. In certainembodiments, the cell or tissue is an N2a cell, a primary neuronalculture or a hippocampal tissue explant.

In one embodiment, the Aβ levels are lowered. In another embodiment, theAβ levels are raised. In another embodiment, the Aβ is Aβ40. In anotherembodiment, the Aβ is Aβ42.

In another embodiment, the modulating results in an increase in theratio of Aβ40 to Aβ42. In another embodiment, the modulating results inan increase in C99.

In another embodiment, the compound binds an enzyme that exhibits anATP-dependent enzymatic activity. In another embodiment, the compoundbinds a molecule that regulates the ATP-enzymatic activity.

In another embodiment, the molecule is a molecule that is allostericallyregulated by ATP.

In another embodiment, the compound is an ATP modulator. In anotherembodiment, the ATP modulator is a selective modulator.

In another embodiment, the ATP modulator competes with ATP for bindingto an ATP-binding site. In another embodiment, the ATP-binding site ispresent on an enzyme that exhibits an ATP-dependent enzymatic activity.In another embodiment, the ATP-binding site is present on a moleculethat regulates the ATP-dependent enzymatic activity.

Binding may be measured under any standard art-known physiologicalconditions, according to methods well known in the art.

In another embodiment, the compound does not affect total cellularlevels of β-amyloid precursor product (APP). In another embodiment, thecompound does not decrease levels of secreted APP (sAPP). In anotherembodiment, the compound increases levels of sAPPα. In anotherembodiment, levels of secreted Aβ are modulated. In another embodiment,the compound does not inhibit Notch-1 cleavage. In another embodiment,the compound does not affect tau phosphorylation.

In another embodiment, the compound crosses the blood-brain barrier.

In another embodiment, the enzymatic activity is a γ-secretase activity.In another embodiment, the compound inhibits γ-secretase activity. Inanother embodiment, the compound is an ATP modulator. In anotherembodiment, the ATP modulator is a selective modulator.

In another embodiment, the compound binds a γ-secretase enzymeexhibiting an ATP-dependent enzymatic activity. In another embodiment,the ATP modulator competes with ATP for binding to an ATP binding site.

In another embodiment, the compound is STI-571 or a blood-brain barrierpermeable variant thereof, or a pharmaceutically acceptable saltthereof. In another embodiment, the compound is a mesylate salt ofSTI-571. In another embodiment, the compound is WGB-BC-15 or ablood-brain barrier permeable variant thereof, or a pharmaceuticallyacceptable salt thereof. In another embodiment, the compound is Compound1 or a blood-brain barrier permeable variant thereof, or apharmaceutically acceptable salt thereof. In another embodiment, thecompound is Compound 2 or a blood-brain barrier permeable variantthereof, or a pharmaceutically acceptable salt thereof. In anotherembodiment, the compound is Compound 3 (PD173956; Bauer et al., 2001,Thromb. Haemost. 85(2):331-40, Maschberger et al., 2000, J. Biol. Chem.275(25):19159-66) or a blood-brain barrier permeable variant thereof, ora pharmaceutically acceptable salt thereof. In another embodiment, thecompound is Compound 4 (PD173952; Dorsey et al., 2002, Leukemia16(9):1589-95) or a blood-brain barrier permeable variant thereof, or apharmaceutically acceptable salt thereof. In another embodiment, thecompound is Compound 5 (PD173958; Dorsey et al., 2002, Leukemia16(9):1589-95; Joseloffet et al., 2002, J. Biol. Chem. 277(14):12318-23)or a blood-brain barrier permeable variant thereof, or apharmaceutically acceptable salt thereof.

In another embodiment, the enzymatic activity is a kinase activity. Inanother embodiment, the compound binds to a kinase enzyme exhibiting anATP-dependent enzymatic activity. In another embodiment, the compoundcompetes with ATP for binding to an ATP-binding site on the kinase. Inanother embodiment, the compound binds to a site other than anATP-binding site on the kinase. In another embodiment, the kinase is atyrosine kinase. In another embodiment, the tyrosine kinase is Ablkinase, BCR-Abl kinase, ARG kinase, src kinase, c-kit orplatelet-derived growth factor receptor. In another embodiment, thekinase is a scrine/threonine kinase, a carbohydrate kinase or a lipidkinase.

In another aspect, the invention provides a method for modulating Aβlevels exhibited by a cell or tissue comprising contacting said cell ortissue with an amount of an ATP modulator sufficient to modulate said Aβlevels.

In one embodiment, the Aβ levels are lowered. In another embodiment, theAβ levels are raised. In another embodiment, the Aβ is A 40. In anotherembodiment, the Aβ is Aβ42.

In another embodiment, the modulating results in an increase in theratio of Aβ40 to Aβ42. In another embodiment, the modulating results inan increase in C99. In another embodiment, the ATP modulator competeswith ATP for binding to an ATP-binding site.

In another embodiment, the ATP-binding site is present on an enzyme thatexhibits an ATP-dependent enzymatic activity. In another embodiment, theATP-binding site is present on a molecule that regulates theATP-dependent enzymatic activity. In another embodiment, the compounddoes not affect total cellular levels of β-amyloid precursor product(APP). In another embodiment, the compound does not decrease levels ofsecreted APP (sAPP). In another embodiment, the compound increaseslevels of sAPPα.

In another embodiment, levels of secreted Aβ are modulated.

In another embodiment, the compound does not inhibit Notch-1 cleavage.In another embodiment, the compound does not affect tau phosphorylation.In another embodiment, the compound crosses the blood-brain barrier. Inanother embodiment, the enzymatic activity is a γ-secretase activity. Inanother embodiment, the compound inhibits γ-secretase activity.

In another embodiment, the compound is an ATP modulator. In anotherembodiment, the ATP modulator is a selective modulator. In anotherembodiment, the compound binds a γ-secretase enzyme exhibiting anATP-dependent enzymatic activity.

In another embodiment, the compound is STI-571 or a blood-brain barrierpermeable variant thereof, or a pharmaceutically acceptable saltthereof. In another embodiment, the compound is a mesylate salt ofSTI-571. In another embodiment, the compound is WGB-BC-15 or ablood-brain barrier permeable variant thereof, or a pharmaceuticallyacceptable salt thereof. In another embodiment, the compound is any ofCompounds 1-5 or a blood-brain barrier permeable variant thereof, or apharmaceutically acceptable salt thereof.

In another embodiment, the enzymatic activity is a kinase activity. Inanother embodiment, the compound binds to a kinase enzyme exhibiting anATP-dependent enzymatic activity. In another embodiment, the compoundcompetes with ATP for binding to an ATP-binding site on the kinase. Inanother embodiment, the compound binds to a site other than anATP-binding site on the kinase. In another embodiment, the kinase is atyrosine kinase. In another embodiment, the tyrosine kinase is Ablkinase, BCR-Abl kinase, ARG kinase, src kinase, c-kit orplatelet-derived growth factor receptor. In another embodiment, thekinase is a serine/threonine kinase, a carbohydrate kinase or a lipidkinase. In another embodiment, the kinase is a serine/threonine kinase,a carbohydrate kinase or a lipid kinase.

In another embodiment, the compound competes with ATP for binding to atarget other than γ-secretase in the metabolic pathway that regulatesthe cleavage of APP to Aβ by γ-secretase. In specific embodiments, thetarget is a kinase (or kinase domain), a protease (e.g., an AAA proteaseof the kind localized to the inner mitochondrial membrane), aphosphatase, or a molecular chaperone molecule (e.g., hsp 60, hsp70,hsp90).

In certain embodiments, γ-secretase activity may reflect assembly,allosteric activation, or stabilization of a protein complex. In certainembodiments, the level of γ-secretase activity is a level of thisprotein complex, and the level of the protein complex can be determinedby measuring the levels of the component proteins in the complex bymethods well known in the art, e.g., by immunoblotting.

In another embodiment, the compound inhibits γ-secretase activity eitherby binding to a site on γ-secretase, or on a component protein ofγ-secretase (as in a γ-secretase multimolecular complex that camcomprise presenilin, nicastrin and APH1), that otherwise can be occupiedby ATP (where ATP-binding is necessary for kinase activity or foraccelerating γ-secretase activity). According to the invention, suchcompounds antagonize ATP. Alternatively, in certain embodiments, thesecompounds may bind to a modulator of γ-secretase activity at an ATPbinding site, wherein said modulator is a factor that is necessary forassembly, stability, trafficking or activation of γ-secretase activityand is antagonized by binding of the compound.

In other embodiments, the level of Aβ is modulated via modulation ofγ-secretase activity.

In one aspect, the invention provides a method for modulating Aβ levelsin a cell or tissue comprising contacting said cell or tissue with anamount of a compound sufficient to modulate said Aβ levels, wherein saidcompound contains the following pharmacophore:

-   -   wherein X is CH—, O, NH or N—CO—.

In one embodiment, said pharmacophore containing compound comprises acompound of Figure I or a pharmaceutically acceptable salt thereof:Figure I

-   -   A is CH or N;    -   B and C are independently CH, N or N⁺—O—;

R¹ is H, SO₂R^(a), (C═O)_(r)O_(s)R^(a);

R², R³, R⁴ and R⁵ are independently H, OH, CHO, CN, halogen,(C═O)_(r)O_(s)(C₁-C₁₀)alkyl, (C═O)_(r)O_(s)(C₂-C₁₀)alkenyl,(C═O)_(r)O_(s)(C₂-C₁₀)alkynyl, (C═O)_(r)O_(s)cycloalkyl,(C═O)_(r)O_(s)cycloalkenyl, (C═O)_(r)O_(s)cycloalkynyl,(C═O)_(r)O_(s)heterocycyl, (C═O)_(r)O_(s)aryl, (C═O)_(r)O_(s)heteroaryl,(C═O)_(r)O_(s)perfluoroalkyl or (C₀-C₆)alkyl-NR^(b)R^(c),

wherein said alkykl, alkenyl, alkynyl, cycyloalkyl, cycloalkenyl,cycloalkynyl, heterocycyl, aryl, heteroaryl and perfluoroalkyl isoptionally substituted with one or more substituents selected from R⁶;

-   -   R⁶ is (C═O)_(r)O_(s)NR^(a)R^(b), (C═O)_(r)O_(s)aryl,        (C═O)_(r)O_(s)heterocycyl, halogen, OH, oxo,        (C═O)_(r)O_(s)(C₁-C₃)perfluoroalkyl, (C═O)_(r)O_(s)(C₁-C₆)alkyl,        CHO, CO₂H, CN, (C₀-C₆)alkyl-NR^(b)R^(c) or        (C₁-C₆)alkyl-heterocycyl,        wherein said alkyl-heterocycyl is optionally substituted with        OH;    -   R^(a) is (C₁-C₆)alkyl, aryl or heterocycyl; and    -   R^(b) and R^(c) independently are H,        (C═O)_(r)O_(s)(C₁-C₁₀)alkyl, SO₂R^(a),        (C═O)_(r)O_(s)heterocycyl, (C═O)_(r)O_(s)aryl,        (C═O)_(r)O_(s)heteroaryl or CO₂R^(a),        wherein r and s independently are 0 or 1 and said alkyl,        heterocycyl, aryl or heteroaryl is optionally substituted with        one or more substituents selected from R⁶.

In another embodiment, A is CH, B is N and C is CH.

In another embodiment, R¹ is H.

In another embodiment, R² is H and R³ is heteroaryl.

In another embodiment, R⁴ is (C═O)_(r)O_(s)(C₁-C₁₀)alkyl and R⁵ is(C₀-C₆)alkyl-NR^(b)R^(c).

In another embodiment, R³ is 3-pyridinyl.

In another embodiment, r is 0, s is 0 and (C₁-C₁₀)alkyl is methyl.

In another embodiment, (C₀-C₆)alkyl is a direct bond (C₀-C₆), R^(b) is Hand R^(c) is (C═O)_(r)O_(s)heteroaryl or (C═O)_(r)O_(s)heterocycyl.

In another embodiment, (C═O)_(r)O_(s)heterocycyl is4-hydroxy-1-piperazino, as illustrated below or a pharmaceuticallyacceptable salt thereof.

In another embodiment, (C═O)_(r)O_(s)heteroaryl is 3-pyrindinyl, asillustrated below or a pharmaceutically acceptable salt thereof.

In another embodiment, said pharmacophore containing compound, comprisesa compound of Figure II or a pharmaceutically acceptable salt thereof:

Figure II

-   -   A, B and C are independently CH, N or N⁺—O—;    -   D is O, S or N—R⁵;    -   R¹ is H, SO₂R^(a), (C═O)_(r)R^(a) or CO₂R^(a);    -   R², R³ and R⁴ are independently H, OH, CHO, CN, halogen,        (C═O)_(r)O_(s)(C₁-C₁₀)alkyl, (C═O)_(r)O_(s)(C₂-C₁₀)alkenyl,        (C═O)_(r)O_(s)(C₂-C₁₀)alkynyl, (C═O)_(r)O_(s)cycloalkyl,        (C═O)_(r)O_(s)cycloalkenyl, (C═O)_(r)O_(s)cycloalkynyl,        (C═O)_(r)O_(s)heterocycyl, (C═O)_(r)O_(s)aryl,        (C═O)_(r)O_(s)heteroaryl, (C═O)_(r)O_(s)perfluoroalkyl or        (C₀-C₆)alkyl-NR^(b)R^(c),        wherein said alkykl, alkenyl, alkynyl, cycyloalkyl,        cycloalkenyl, cycloalkynyl, heterocycyl, aryl, heteroaryl and        perfluoroalkyl is optionally substituted with one or more        substituents selected from R⁶;    -   R⁵ is H, aryl or (C₁-C₆)alkyl;    -   R⁶ is (C═O)_(r)O_(s)NR^(a)R^(b), (C═O)_(r)O_(s)aryl,        (C═O)_(r)O_(s)heterocycyl, halogen, OH, oxo,        (C═O)_(r)O_(s)(C₁-C₃)perfluoroalkyl, (C═O)_(r)O_(s)(C₁-C₆)alkyl,        CHO, CO₂H, CN, (C₀-C₆)alkyl-NR^(b)R^(c) or        (C₁-C₆)alkyl-heterocycyl;    -   R^(a) is (C₁-C₆)alkyl, aryl or heterocycyl; and    -   R^(b) and R^(c) independently are H,        (C═O)_(r)O_(s)(C₁-C₁₀)alkyl, SO₂R^(a),        (C═O)_(r)O_(s)heterocycyl, (C═O)_(r)O_(s)aryl,        (C═O)_(r)O_(s)heteroaryl or CO₂R,        wherein r and s independently are 0 or 1 and said alkyl,        heterocycyl, aryl or heteroaryl is optionally substituted with        one or more substituents selected from R⁶.

In another embodiment, R¹ is H.

In another embodiment, R² is H and R³ is (CO—C₆)alkyl-NR^(b)R^(c).

In another embodiment, R⁴ is heteroaryl.

In another embodiment, said pharmacophore containing compound, comprisesa compound of Figure III or a pharmaceutically acceptable salt thereof:

Figure III

-   -   A is aryl or heteroaryl,        wherein said aryl or heteroaryl is optionally substituted with        one or more substituents selected from R³;    -   X is NH, N-acyl, O or S;    -   R¹ and R² are independently H, OH, CHO, CN, halogen,        (C═O)_(r)O_(s)(C₁-C₁₀)alkyl, (C═O)_(r)O_(s)(C₂-C₁₀)alkenyl,        (C═O)_(r)O_(s)(C₂-C₁₀)alkynyl, (C═O)_(r)O_(s)cycloalkyl,        (C═O)_(r)O_(s)cycloalkenyl, (C═O)_(r)O_(s)cycloalkynyl,        (C═O)_(r)O_(s)heterocycyl, (C═O)_(r)O_(s)aryl,        (C═O)_(r)O_(s)heteroaryl, (C═O)_(r)O_(s)perfluoroalkyl or        (C₀-C₆)alkyl-NR^(a)R^(b),        wherein said alkykl, alkenyl, alkynyl, cycyloalkyl,        cycloalkenyl, cycloalkynyl, heterocycyl, aryl, heteroaryl and        perfluoroalkyl is optionally substituted with one or more        substituents selected from R³;    -   R³ is (C═O)_(r)O_(s)NR^(a)R^(b), (C═O)_(r)O_(s)aryl,        (C═O)_(r)O_(s)heterocycyl, halogen, OH, oxo,        (C═O)_(r)O_(s)(C₁-C₃)perfluoroalkyl,        (C═O)_(r)O_(s)S_(t)(C₁-C₆)alkyl, CHO, CO₂H, CN,        (C₀-C₆)alkyl-NR^(a)R^(b) or (C₁-C₆)alkyl-heterocycyl;    -   R^(a) and R^(b) independently are H,        (C═O)_(r)O_(s)(C₁-C₁₀)alkyl, SO₂R¹, (C═O)_(r)O_(s)heterocycyl,        (C═O)_(r)O_(s)aryl, (C═O)_(r)O_(s)heteroaryl or CO₂R¹,        wherein r, s and t independently are 0 or 1 and said alkyl,        heterocycyl, aryl or heteroaryl is optionally substituted with        one or more substituents selected from R³.

In another embodiment, A is aryl.

In another embodiment, the aryl is 2,5-dichlorophenyl.

In another embodiment, R¹ is (C_(O-)C₆)alkyl-NR^(a)R^(b) and R² is(C═O)_(r)O_(s)(C₁-C₁₀)alkyl.

In another embodiment, (C═O)_(r)O_(s)(C₁-C₁₀)alkyl is methyl.

In another embodiment, (C_(O-)C₆)alkyl-NR^(a)R^(b) is

or a pharmaceutically acceptable salt thereof.

In another aspect, the invention provides a pharmaceutical compositionuseful for modulating Aβ level in a cell or tissue, comprising:

-   -   an amount of the compound described hereinabove sufficient to        modulate said Aβ level; and    -   a pharmaceutically acceptable excipient or carrier.

In one embodiment, the pharmaceutical composition is useful formodulating Aβ levels exhibited by a cell or tissue.

In another embodiment, the compound is present in an amount sufficientto modulate said Aβ levels.

Compounds according to Figure I may be prepared by various processesknown to one of skill in the art. In particular, compounds according toFigure I, wherein A is CH, B is N, C is CH and R¹ is H may be preparedby the following process:

-   -   a) a compound according to Figure IV        Figure IV

wherein Y and Z constitute (C₁-C₆)alkyl groups and R² and R³ are asabove-defined is reacted with a compound according to Figure VFigure V

wherein R⁴ and R⁵ are as above-defined.

In certain embodiments, the compound is a compound identified by themethods of the invention, wherein the compound modulates γ-secretaseactivity, e.g., ATP-dependent γ-secretase activity, and whereinmodulation of γ-secretase activity results in an alteration in the levelof Aβ exhibited by a cell or tissue. γ-secretase activity is well knownin the art, and includes cleavage of APP at the γ-secretase cleavageregion.

In one embodiment, a method is provided for modulating γ-secretaseactivity in cells or tissues of interest in vitro.

In another embodiment, γ-secretase activity in cells or tissues ofinterest is modulated in situ or in vivo. The in vitro, in situ and invivo applications may include, but are not limited to modulatingactivity in any of the cells or tissues disclosed hereinabove.

According to the invention, a substance “exhibited by a cell or tissue”encompasses a substance that it produced, sequestered, taken up orreleased by, the cell or tissue and that either remains within the cell(or tissue), i.e., intracellularly, or is secreted or released by thecell (or tissue) (extracellularly).

5.1.1. Therapeutic Methods

The present invention provides methods for prevention, treatment, e.g.,management, of an Aβ-related disorder, or amelioration of a symptom ofan Aβ-related disorder such as Alzheimer's disease. It is understoodthat the methods described herein in the context of treating and/orameliorating a symptom can also routinely be utilized as part of aprevention protocol.

The invention also provides a method of treating, or ameliorating asymptom of, an Aβ-related disorder comprising administering to a subjectin need of such treating or ameliorating an amount of a compoundsufficient to reduce Aβ levels in the subject, wherein the compoundmodulates an ATP-dependent enzymatic activity, such that the Aβ-relateddisorder is treated or a symptom of the Aβ related disorder isameliorated.

In one embodiment, the Aβ related disorder is Alzheimer's disease. Inanother embodiment, progression of the Aβ related disorder is slowed. Inanother embodiment, progression of the Aβ related disorder is reversed.

In another embodiment, the subject is a human subject. In a specificembodiment, the subject is a subject at risk for a familial form ofAlzheimer's disease.

In another embodiment, the ATP-dependent enzymatic activity is lowered.In another embodiment, the compound crosses the blood-brain barrier. Inanother embodiment, the compound is administered orally. As used herein,this can mean either concurrent or sequential administration.

In another embodiment, the compound is administered with a NSAID. In aspecific embodiment, the NSAID is sulindac sulfide, flufenamic acid,ibuprofen, flurbiprofen, fenoprofen, mefenamic acid, indomethacin,carprofen, meclofenamic acid.

In another embodiment, the compound is administered with an antioxidant.In a specific embodiment, the antioxidant is selected from the groupconsisting of vitamin E, vitamin C, curcumin, and Gingko biloba.

In another embodiment, the compound is administered with anacetylcholinesterase inhibitor.

In another embodiment, the compound is an ATP modulator. In anotherembodiment, the compound is ATP modulator is a selective modulator.

In another embodiment, the enzymatic activity is a γ-secretase activity.In another embodiment, the compound binds a γ-secretase enzymeexhibiting an ATP-dependent enzymatic activity.

In another embodiment, the ATP modulator competes with ATP for bindingto an ATP binding site.

In one embodiment, the compound is STI-571 or a blood-brain barrierpermeable variant thereof, or a pharmaceutically acceptable saltthereof. In another embodiment, the compound is a mesylate salt ofSTI-571. In another embodiment, the compound is WGB-BC-15 or ablood-brain barrier permeable variant thereof, or a pharmaceuticallyacceptable salt thereof. In another embodiment, the compound is any ofCompounds 1-5 or a blood-brain barrier permeable variant thereof, or apharmaceutically acceptable salt thereof.

In one embodiment, the enzymatic activity is a kinase activity. In oneembodiment, the compound binds to a kinase enzyme exhibiting anATP-dependent enzymatic activity. In one embodiment, the compoundcompetes with ATP for binding to an ATP-binding site on the kinase. Inanother embodiment, the compound binds to a site other than anATP-binding site on the kinase.

In a specific embodiment, the kinase is a tyrosine kinase. In anotherembodiment, the tyrosine kinase is Abl kinase, BCR-Abl kinase, ARGkinase, src kinase, c-kit, platelet-derived growth factor receptor.

In a specific embodiment, the kinase is a serine/threonine kinase, acarbohydrate kinase or a lipid kinase.

In another embodiment, the enzymatic activity is activity of a protease(e.g., AAA protease of the kind localized to the inner mitochondrialmembrane), phosphatase, or a molecular chaperone molecule (e.g., hsp 60,hsp70, hsp90).

The invention also provides a method of treating, or ameliorating asymptom of, an Aβ-related disorder comprising administering to a subjectin need of such treating or ameliorating an amount of an ATP modulatorsufficient to reduce Aβ levels in the subject, such that the Aβ-relateddisorder is treated or a symptom of the Aβ related disorder isameliorated.

In one embodiment, the Aβ related disorder is Alzheimer's disease. Inone embodiment, the subject is a human subject.

In one embodiment, the subject is a subject at risk for a familial formof Alzheimer's disease.

In another embodiment, the ATP modulator crosses the blood-brainbarrier.

In another embodiment, the ATP modulator is administered orally. Inanother embodiment, the ATP modulator is administered with a NSAID. Inanother embodiment, the NSAID is sulindac sulfide, flufenamic acid,ibuprofen, flurbiprofen, fenoprofen, mefenamic acid, indomethacin,carprofen, meclofenamic acid.

In another embodiment, the ATP modulator is administered with anantioxidant. In another embodiment, the antioxidant is selected from thegroup consisting of vitamin E, vitamin C, curcumin, and Gingko biloba.

In another embodiment, the ATP modulator is administered with anacetylcholinesterase inhibitor.

In another embodiment, the ATP modulator is a selective modulator.

In another embodiment, the enzymatic activity is a γ-secretase activity.

In one embodiment, the compound binds a γ-secretase enzyme exhibiting anATP-dependent enzymatic activity. In another embodiment, the ATPmodulator competes with ATP for binding to an ATP binding site.

In one embodiment, the compound is STI-571 or a blood-brain barrierpermeable variant thereof, or a pharmaceutically acceptable saltthereof. In another embodiment, the compound is a mesylate salt ofSTI-571. In another embodiment, the compound is WGB-BC-15 or ablood-brain barrier permeable variant thereof, or a pharmaceuticallyacceptable salt thereof. In another embodiment, the compound is any ofCompounds 1-5 or a blood-brain barrier permeable variant thereof, or apharmaceutically acceptable salt thereof.

In one embodiment, the enzymatic activity is a kinase activity. In oneembodiment, the compound binds to a kinase enzyme exhibiting anATP-dependent enzymatic activity. In one embodiment, the compoundcompetes with ATP for binding to an ATP-binding site on the kinase. Inanother embodiment, the compound binds to a site other than anATP-binding site on the kinase.

In a specific embodiment, the kinase is a tyrosine kinase. In anotherembodiment, the tyrosine kinase is Abl kinase, BCR-Abl kinase, ARGkinase, src kinase, c-kit, platelet-derived growth factor receptor.

In a specific embodiment, the kinase is a serine/threonine kinase, acarbohydrate kinase or a lipid kinase.

In another embodiment, the enzymatic activity is activity of a protease(e.g., AAA protease of the kind localized to the inner mitochondrialmembrane), phosphatase, or a molecular chaperone molecule (e.g., hsp 60,hsp70, hsp90).

The invention also provides a method of treating, or ameliorating asymptom of, an Aβ-related disorder comprising administering to a subjectin need of such treating or ameliorating an amount of a compoundsufficient to reduce Aβ levels in the subject, such that the Aβ-relateddisorder is treated or a symptom of the Aβ-related disorder isameliorated, wherein the compound contains the following pharmacophore:

wherein X is CH—, O, NH or N—CO—.

In one embodiment, said pharmacophore containing compound comprises acompound of Figure I or a pharmaceutically acceptable salt thereof:

Figure I

-   -   A is CH or N;    -   B and C are independently CH, N or N⁺—O—;    -   R¹ is H, SO₂R^(a), (C═O)_(r)O_(s)R^(a);    -   R², R³, R⁴ and R⁵ are independently H, OH, CHO, CN, halogen,        (C═O)_(r)O_(s)(C₁-C₁₀)alkyl, (C═O)_(r)O_(s)(C₂-C₁₀)alkenyl,        (C═O)_(r)O_(s)(C₂-C₁₀)alkynyl, (C═O)_(r)O_(s)cycloalkyl,        (C═O)_(r)O_(s)cycloalkenyl, (C═O)_(r)O_(s)cycloalkynyl,        (C═O)_(r)O_(s)heterocycyl, (C═O)_(r)O_(s)aryl,        (C═O)_(r)O_(s)heteroaryl, (C═O)_(r)O_(s)perfluoroalkyl or        (C₀-C₆)alkyl-NR^(b)R^(c),        wherein said alkykl, alkenyl, alkynyl, cycyloalkyl,        cycloalkenyl, cycloalkynyl, heterocycyl, aryl, heteroaryl and        perfluoroalkyl is optionally substituted with one or more        substituents selected from R⁶;    -   R⁶ is (C═O)_(r)O_(s)NR^(a)R^(b), (C═O)_(r)O_(s)aryl,        (C═O)_(r)O_(s)heterocycyl, halogen, OH, oxo,        (C═O)_(r)O_(s)(C₁-C₃)perfluoroalkyl, (C═O)_(r)O_(s)(C₁-C₆)alkyl,        CHO, CO₂H, CN, (C₀-C₆)alkyl-NR^(b)R^(c) or        (C₁-C₆)alkyl-heterocycyl,        wherein said alkyl-heterocycyl is optionally substituted with        OH;    -   R¹ is (C₁-C₆)alkyl, aryl or heterocycyl; and    -   R^(b) and R^(c) independently are H,        (C═O)_(r)O_(s)(C₁-C₁₀)alkyl, SO₂R^(a),        (C═O)_(r)O_(s)heterocycyl, (C═O)_(r)O_(s)aryl,        (C═O)_(r)O_(s)heteroaryl or CO₂R^(a),        wherein r and s independently are 0 or 1 and said alkyl,        heterocycyl, aryl or heteroaryl is optionally substituted with        one or more substituents selected from R⁶.

In another embodiment, A is CH, B is N and C is CH.

In another embodiment, R¹ is H.

In another embodiment, R² is H and R³ is heteroaryl.

In another embodiment, R⁴ is (C═O)_(r)O_(s)(C₁-C₁₀)alkyl and R⁵ is(C₀-C₆)alkyl-NR^(b)R^(c).

In another embodiment, R³ is 3-pyridinyl.

In another embodiment, r is 0, s is 0 and (C₁-C₁₀)alkyl is methyl.

In another embodiment, (C₀-C₆)alkyl is a direct bond (C₀), R^(b) is Hand R^(c) is (C═O)_(r)O_(s)heteroaryl or (C═O)_(r)O_(s)heterocycyl.

In another embodiment, (C═O)_(r)O_(s)heterocycyl is4-hydroxy-1-piperazino, as illustrated below or a pharmaceuticallyacceptable salt thereof.

In another embodiment, the pharmacophore containing compound comprises acompound of Figure II or a pharmaceutically acceptable salt thereof:

Figure II

-   -   A, B and C are independently CH, N or N⁺—O—;    -   D is O, S or N—R⁵;    -   R¹ is H, SO₂R^(a), (C═O)_(r)R^(a) or CO₂R^(a);    -   R², R³ and R⁴ are independently H, OH, CHO, CN, halogen,        (C═O)_(r)O_(s)(C₁-C₁₀)alkyl, (C═O)_(r)O_(s)(C₂-C₁₀)alkenyl,        (C═O)_(r)O_(s)(C₂-C₁₀)alkynyl, (C═O)_(r)O_(s)cycloalkyl,        (C═O)_(r)O_(s)cycloalkenyl, (C═O)_(r)O_(s)cycloalkynyl,        (C═O)_(r)O_(s)heterocycyl, (C═O)_(r)O_(s)aryl,        (C═O)_(r)O_(s)heteroaryl, (C═O)_(r)O_(s)perfluoroalkyl or        (C₀-C₆)alkyl-NR^(b)R^(c),        wherein said alkykl, alkenyl, alkynyl, cycyloalkyl,        cycloalkenyl, cycloalkynyl, heterocycyl, aryl, heteroaryl and        perfluoroalkyl is optionally substituted with one or more        substituents selected from R⁶;    -   R⁵ is H, aryl or (C₁-C₆)alkyl;    -   R⁶ is (C═O)_(r)O_(s)NR^(a)R^(b), (C═O)_(r)O_(s)aryl,        (C═O)_(r)O_(s)heterocycyl, halogen, OH, oxo,        (C═O)_(r)O_(s)(C₁-C₃)perfluoroalkyl, (C═O)_(r)O_(s)(C₁-C₆)alkyl,        CHO, CO₂H, CN, (C₀-C₆)alkyl-NR^(b)R^(c) or        (C₁-C₆)alkyl-heterocycyl;    -   R^(a) is (C₁-C₆)alkyl, aryl or heterocycyl; and    -   R^(b) and R^(c) independently are H,        (C═O)_(r)O_(s)(C₁-C₁₀)alkyl, SO₂R^(a),        (C═O)_(r)O_(s)heterocycyl, (C═O)_(r)O_(s)aryl,        (C═O)_(r)O_(s)heteroaryl or CO₂R^(a), wherein r and s        independently are 0 or 1 and said alkyl, heterocycyl, aryl or        heteroaryl is optionally substituted with one or more        substituents selected from R⁶.

In another embodiment, R¹ is H.

In another embodiment, R² is H and R³ is (C₀-C₆)alkyl-NR^(b)R^(c).

In another embodiment, R⁴ is heteroaryl.

In another embodiment, said pharmacophore containing compound, comprisesa compound of Figure III or a pharmaceutically acceptable salt thereof:

Figure III

-   -   A is aryl or heteroaryl,        wherein said aryl or heteroaryl is optionally substituted with        one or more substituents selected from R³;    -   X is NH, N-acyl, O or S;    -   R¹ and R² are independently H, OH, CHO, CN, halogen,        (C═O)_(r)O_(s)(C₁-C₁₀)alkyl, (C═O)_(r)O_(s)(C2-C₁₀)alkenyl,        (C═O)_(r)O_(s)(C2-C₁₀)alkynyl, (C═O)_(r)O_(s)cycloalkyl,        (C═O)_(r)O_(s)cycloalkenyl, (C═O)_(r)O_(s)cycloalkynyl,        (C═O)_(r)O_(s)heterocycyl, (C═O)_(r)O_(s)aryl,        (C═O)_(r)O_(s)heteroaryl, (C═O)_(r)O_(s)perfluoroalkyl or        (C₀-C₆)alkyl-NR^(a)R^(b),        wherein said alkykl, alkenyl, alkynyl, cycyloalkyl,        cycloalkenyl, cycloalkynyl, heterocycyl, aryl, heteroaryl and        perfluoroalkyl is optionally substituted with one or more        substituents selected from R³;    -   R³ is (C═O)_(r)O_(s)NR^(a)R^(b), (C═O)_(r)O_(s)aryl,        (C═O)_(r)O_(s)heterocycyl, halogen, OH, oxo,        (C═O)_(r)O_(s)(C₁-C₃)perfluoroalkyl,        (C═O)_(r)O_(s)S_(t)(C₁-C₆)alkyl, CHO, CO₂H, CN,        (C₀-C₆)alkyl-NR^(a)R^(b) or (C₁-C₆)alky-heterocycyl;    -   R^(a) and R^(b) independently are H,        (C═O)_(r)O_(s)(C₁-C₁₀)alkyl, SO₂R¹, (C═O)_(r)O_(s)heterocycyl,        (C═O)_(r)O_(s)aryl, (C═O)_(r)O_(s)heteroaryl or CO₂R^(a),        wherein r, s and t independently are 0 or 1 and said alkyl,        heterocycyl, aryl or heteroaryl is optionally substituted with        one or more substituents selected from R³.

In another embodiment, A is aryl.

In another embodiment, aryl is 2,5-dichlorophenyl.

In another embodiment, R¹ is (C₀-C₆)alkyl-NR^(a)R^(b) and R² is(C═O)_(r)O_(s)(C₁-C₁₀)alkyl.

In another embodiment, (C═O)_(r)O_(s)(C₁-C₁₀)alkyl is methyl.

In another embodiment, (C₀-C₆)alkyl-NR^(a)R^(b) is

or a pharmaceutically acceptable salt thereof.

In another embodiment, the agent is administered orally.

In one embodiment, the compound is administered with a NSAID. In anotherembodiment, the NSAID is sulindac sulfide, flufenamic acid, ibuprofen,flurbiprofen, fenoprofen, mefenamic acid, indomethacin, carprofen,meclofenamic acid.

In one embodiment, the compound is administered with an antioxidant. Ina specific embodiment, the antioxidant is selected from the groupconsisting of vitamin E, vitamin C, curcumin, and Gingko biloba.

In another embodiment, the compound is administered with anacetylcholinesterase inhibitor.

5.1.2. Modulating Compounds

5.1.2.1. Compounds That Modulate Aβ Levels

The present invention also provides compositions for modulating Aβlevels exhibited by a cell or tissue, including, but not limited to thefollowing compounds (or agents) disclosed hereinbelow. The inventionalso provides compositions for modulating the level Aβ via modulation ofthe activity of γ-secretase, including, but not limited to the followingagents, drugs, compounds or small molecules disclosed hereinbelow.

In one embodiment, the invention provides a composition for modulatingAβ level exhibited by a cell or tissue comprising a modulator ofATP-dependent γ-secretase activity and a pharmaceutically acceptablevehicle. In one aspect of this embodiment, the Aβ level is lowered. Inanother aspect, the Aβ level is raised. In another aspect, the Aβ isAβ40. In another aspect, the Aβ is Aβ42. In another aspect, themodulator binds to a kinase. In another aspect, the modulator competeswith ATP for binding to the ATP-binding site on a kinase. In anotheraspect, the compound binds to a site other than an ATP-binding site onthe kinase. In another aspect, the compound is an ATP modulator. Inanother aspect, the kinase may be, e.g., Abl kinase, ARG kinase, BCR-Ablkinase, src kinase, c-kit or platelet-derived growth factor receptor. Inanother aspect, the modulator does not modulate Abl kinase activity. Inanother aspect, the modulator is an inhibitor of ATP-dependent enzymaticactivity. In another aspect, the modulator does not inhibit Notch-1cleavage. In another aspect, the modulator can cross the blood-brainbarrier. In another aspect, the inhibitor is a selective inhibitor. Inanother aspect, the selective inhibitor is STI-571, WGB-BC-415, Compound1, Compound 2, Compound 3, Compound 4, Compound 5 or a blood-brainbarrier permeable variant thereof.

Among the preferred compounds are those that are ATP modulators.

In certain embodiments, the ATP modulator, such as an ATP inhibitor, mayinclude, but not be limited to, a phenylaminopyrimidine tyrosine kinaseinhibitor, e.g., 2-phenylaminopyrimidine, a pyrimidinyl pyridionetyrosine kinase inhibitor, 2-(Purin-9-yl)-tetrahydrofuran-3,4-diolderivatives; pyridoxine and pyridoxal analogues; N-6 heterocyclic8-modified adenosine derivatives; N-6 heterocyclic 5′-modified adenosinederivatives; allosteric inhibitors of pyruvate kinase;8-phenylxanthines, 8-cycloalkylxantines or 8-substituted xanthinederivatives; N-6 substituted adenosine-5′-uronamides; purine,pyrrolo[2,3,d]pyrimidine and pyrazolo[3,4,d]pyrimidine nucleosideanalogs.

In another embodiment, the compound, e.g., ATP modulator, may include,but not be limited to, the following compounds:

Heterocyclic-hydroxyimino-fluorene nuclei compounds; 3-anilinomethyleneoxindoles; 3-(4′-bromobenzylindenyl)-2-indolinone analogues;indeno[1,2,c]-naphthol[1,2,c] and benzo[6,7]cyclohepta[1,2,c]pyrazolederivatives; 3′-epimeric k-252a derivatives; quinazolines;3-cyano-[1,7], [1,5] and [1,8]-napthyridine analogues;N-[4-(3-chloro-4-fluoro-phenylamino)-7-(3-morpholin4-yl-propoxy)-quinazolin-6-yl]-acrylamide;pyrimidine derivatives; benzimidazoles; bicyclic heteroaromaticcompounds; pyrrolopyrimidines; quinoline and quinoxaline derivatives;indolinones; 2-pyrimidineamine derivatives; substitutedpyrido[3,2,d]pyrimidines; fused polycyclic 2-aminopyrimidinederivatives; bicyclic 4-aralkylaminopyrimidine derivatives;N-7-heterocycyl pyrrolo[2,3,d]pyrimidines; 3-cyano quinolinederivatives; pyrazole derivatives; pyrimido[5,4,d]pyrimidines;4-anilinoquinazoline derivatives; 6-aryl napthyridines; N-oxides ofamino containing pyrido[2,3,d]pyrimidines; 5-aminopyrazoles;5,10-dihydropyrimido[4,5,b]quinolin-4(1H)-one; quinolymethylen-oxindoleanalogues; acrylonitrile-sulfonamide derivatives;3-(4′-dimethylaminobenzylidenyl)-2-indolines;3-(2′-alkoxybenzylidenyl)₂-indolines;3-(4′-bromobenzylidenyl)-2-indolines; benzylidene-Z-indoline compounds;4,6-dianilino-pyrimidine derivatives; substitutedindolylmethylene-oxindole analogues; hydrosoluble 3-arylidene-2-oxindoleanalogues; 3-(2′-halobenzylidenyl)-2-indolinone compounds;3-heteroaryl-2-indolinone compounds; benzoylethylene derivatives; ureaand thiourea-type compounds; benzopyran derivatives;pyrido[2,3,d]pyrimidines; 6-aryl-pyrido[2,3,d]pyrimidines andnaphthyridines; substituted 3-arylidene-7-azaoxindole compounds; thienylcompounds; aryl and heteroaryl quinazoline compounds; arylidene andheteroarylidene oxindole derivatives; N-substituted-beta-aryl- andbeta-heteroaryl-alpha-cyanoacrylamide derivatives; 2-iminochromenederivatives; 4-aminopyrrolo[2,3,d]pyrimidines;4-aminopyrazolo(3,4,d)pyrimidine derivatives;4-aminopyrazolo(3,4,d)pyridine derivatives;3-(cycloalkanoheteroarylidenyl)-2-indolinones; isoxazole-4-carboxamidecompounds; 3-(cycloalkanoheteroarylidenyl)-2-indolinones; substitutedphenylacrylonitrile compounds; benzylidene-Z-indoline compounds;3-(2′-halobenzylidenyl)-2-indolinone compounds; benzopyran compounds; or4-aminopyrimidines; isoxazole compounds. Such compounds can, forexample, be utilized as kinase modulators, e.g., inhibitors.

Appropriate compounds can also include, for example, STI-571, mesylatesalt of STI-571 (imatinib mesylate, GLEEVEC™, Novartis Pharmaceuticals),and congeners thereof, members of the 2-phenylaminopyrimidine class ofcompounds, such as Compounds 1-5, as well as compounds that compete forthe ATP binding sites of Bcr-Abl, Abl kinase, or c-kit, platelet-derivedgrowth factor receptors (PDGFR), and on any other kinases known or latershown to be inhibited by STI-571, or a related compound, such as thepyridione tyrosine kinase inhibitor class, would be candidates for usein the present invention.

In another embodiment, agents of the invention inhibit γ-secretaseactivity either by binding to a site on γ-secretase, or on a componentprotein of γ-secretase.

Still further, compounds that can be used as part of the methods of theinvention include, but are not limited to, compounds that contain thefollowing pharmacophore:

-   -   wherein X is CH—, O, NH or N—CO—.

In one embodiment, said pharmacophore containing compound comprises acompound of Figure I or a pharmaceutically acceptable salt thereof:Figure I

-   -   A is CH or N;    -   B and C are independently CH, N or N⁺—O—;    -   R¹ is H, SO₂R^(a), (C═O)_(r)O_(s)R^(a);    -   R², R³, R⁴ and R⁵ are independently H, OH, CHO, CN, halogen,        (C═O)_(r)O_(s)(C₁-C₁₀)alkyl, (C═O)_(r)O_(s)(C₂-C₁₀)alkenyl,        (C═O)_(r)O_(s)(C₂-C₁₀)alkynyl, (C═O)_(r)O_(s)cycloalkyl,        (C═O)_(r)O_(s)cycloalkenyl, (C═O)_(r)O_(s)cycloalkynyl,        (C═O)_(r)O_(s)heterocycyl, (C═O)_(r)O_(s)aryl,        (C═O)_(r)O_(s)heteroaryl, (C═O)_(r)O_(s)perfluoroalkyl or        (C₀-C₆)alkyl-NR^(b)R^(c),        wherein said alkykl, alkenyl, alkynyl, cycyloalkyl,        cycloalkenyl, cycloalkynyl, heterocycyl, aryl, heteroaryl and        perfluoroalkyl is optionally substituted with one or more        substituents selected from R⁶;    -   R⁶ is (C═O)_(r)O_(s)NR^(a)R^(b), (C═O)_(r)O_(s)aryl,        (C═O)_(r)O_(s)heterocycyl, halogen, OH, oxo,        (C═O)_(n)O_(s)(C₁-C₃)perfluoroalkyl, (C═O)_(r)O_(s)(C₁-C₆)alkyl,        CHO, CO₂H, CN, (C₀-C₆)alkyl-NR^(b)R^(c) or        (C₁-C₆)alkyl-heterocycyl,        wherein said alkyl-heterocycyl is optionally substituted with        OH;    -   R^(a) is (C₁-C₆)alkyl, aryl or heterocycyl; and    -   R^(b) and R^(c) independently are H,        (C═O)_(r)O_(s)(C₁-C₁₀)alkyl, SO₂R^(a),        (C═O)_(r)O_(s)heterocycyl, (C═O)_(r)O_(s)aryl,        (C═O)_(r)O_(s)heteroaryl or CO₂R^(a),        wherein r and s independently are 0 or 1 and said alkyl,        heterocycyl, aryl or heteroaryl is optionally substituted with        one or more substituents selected from R⁶.

In another embodiment, A is CH, B is N and C is CH.

In another embodiment, R¹ is H.

In another embodiment, R² is H and R³ is heteroaryl.

In another embodiment, R⁴ is (C═O)_(r)O_(s)(C₁-C₁₀)alkyl and R⁵ is(C₀-C₆)alkyl-NR^(b)R^(c).

In another embodiment, R³ is 3-pyridinyl.

In another embodiment, r is 0, s is 0 and (C₁-C₁₀)alkyl is methyl.

In another embodiment, (C₀-C₆)alkyl is a direct bond (C₀), R^(b) is Hand R^(c) is (C═O)_(r)O_(s)heteroaryl or (C═O)_(r)O_(s)heterocycyl.

In another embodiment, (C═O)_(r)O_(s)heterocycyl is4-hydroxy-1-piperazino, as illustrated below or a pharmaceuticallyacceptable salt thereof.

In another embodiment, (C═O)_(r)O_(s)heteroaryl is 3-pyrindinyl, asillustrated below or a pharmaceutically acceptable salt thereof.

In another embodiment, said pharmacophore containing compound, comprisesa compound of Figure II or a pharmaceutically acceptable salt thereof:

Figure II

-   -   A, B and C are independently CH, N or N⁺—O—;    -   D is O, S or N—R⁵;    -   R¹ is H, SO₂R^(a), (C═O)_(r)R^(a) or CO₂R^(a);    -   R², R³ and R⁴ are independently H, OH, CHO, CN, halogen,        (C═O)_(r)O_(s)(C₁-C₁₀)alkyl, (C═O)_(r)O_(s)(C₂-C₁₀)alkenyl,        (C═O)_(r)O_(s)(C₂-C₁₀)alkynyl, (C═O)_(r)O_(s)cycloalkyl,        (C═O)_(r)O_(s)cycloalkenyl, (C═O)_(r)O_(s)cycloalkynyl,        (C═O)_(r)O_(s)heterocycyl, (C═O)_(r)O_(s)aryl,        (C═O)_(r)O_(s)heteroaryl, (C═O)_(r)O_(s)perfluoroalkyl or        (C₀-C₆)alkyl-NR^(b)R^(c), wherein said alkykl, alkenyl, alkynyl,        cycyloalkyl, cycloalkenyl, cycloalkynyl, heterocycyl, aryl,        heteroaryl and perfluoroalkyl is optionally substituted with one        or more substituents selected from R⁶;    -   R⁵ is H, aryl or (C₁-C₆)alkyl;    -   R⁶ is (C═O)_(r)O_(s)NR^(a)R^(b), (C═O)_(r)O_(s)aryl,        (C═O)_(r)O_(s)heterocycyl, halogen, OH, oxo,        (C═O)_(r)O_(s)(C₁-C₃)perfluoroalkyl, (C═O)_(r)O_(s)(C₁-C₆)alkyl,        CHO, CO₂H, CN, (C₀-C₆)alkyl-NR^(b)R^(c) or        (C₁-C₆)alkyl-heterocycyl;    -   R^(a) is (C₁-C₆)alkyl, aryl or heterocycyl; and    -   R^(b) and R^(c) independently are H, (C═O)_(r)O_(s)(C₁-C₀)alkyl,        SO₂R^(a), (C═O)_(r)O_(s)heterocycyl, (C═O)_(r)O_(s)aryl,        (C═O)_(r)O_(s)heteroaryl or CO₂R^(a),        wherein r and s independently are 0 or 1 and said alkyl,        heterocycyl, aryl or heteroaryl is optionally substituted with        one or more substituents selected from R⁶.

In another embodiment, R¹ is H.

In another embodiment, R² is H and R³ is (C₀-C₆)alkyl-NR^(b)R^(c).

In another embodiment, R⁴ is heteroaryl.

In another embodiment, said pharmacophore containing compound, comprisesa compound of Figure III or a pharmaceutically acceptable salt thereof:

Figure III

-   -   A is aryl or heteroaryl,        wherein said aryl or heteroaryl is optionally substituted with        one or more substituents selected from R³;    -   X is NH, N-acyl, O or S;    -   R¹ and R² are independently H, OH, CHO, CN, halogen,        (C═O)_(r)O_(s)(C₁-C₁₀)alkyl, (C═O)_(r)O_(s)(C₂-C₁₀)alkenyl,        (C═O)_(r)O_(s)(C₂-C₁₀)alkynyl, (C═O)_(r)O_(s)cycloalkyl,        (C═O)_(r)O_(s)cycloalkenyl, (C═O)_(r)O_(s)cycloalkynyl,        (C═O)_(r)O_(s)heterocycyl, (C═O)_(r)O_(s)aryl,        (C═O)_(r)O_(s)heteroaryl, (C═O)_(r)O_(s)perfluoroalkyl or        (C₀-C₆)alkyl-NR^(a)R^(b),        wherein said alkykl, alkenyl, alkynyl, cycyloalkyl,        cycloalkenyl, cycloalkynyl, heterocycyl, aryl, heteroaryl and        perfluoroalkyl is optionally substituted with one or more        substituents selected from R³;    -   R³ is (C═O)_(r)O_(s)NR^(a)R^(b), (C═O)_(r)O_(s)aryl,        (C═O)_(r)O_(s)heterocycyl, halogen, OH, oxo,        (C═O)_(r)O_(s)(C₁-C₃)perfluoroalkyl,        (C═O)_(r)O_(s)S_(t)(C₁-C₆)alkyl, CHO, CO₂H, CN,        (C₀-C₆)alkyl-NR^(a)R^(b) or (C₁-C₆)alkyl-heterocycyl;    -   R^(a) and R^(b) independently are H,        (C═O)_(r)O_(s)(C₁-C₁₀)alkyl, SO₂R^(a),        (C═O)_(r)O_(s)heterocycyl, (C═O)_(r)O_(s)aryl,        (C═O)_(r)O_(s)heteroaryl or CO₂R¹,        wherein r, s and t independently are 0 or 1 and said alkyl,        heterocycyl, aryl or heteroaryl is optionally substituted with        one or more substituents selected from R³.

In another embodiment, A is aryl.

In another embodiment, the aryl is 2,5-dichlorophenyl.

In another embodiment, R¹ is (C₀-C₆)alkyl-NR^(a)R^(b) and R² is(C═O)_(r)O_(s)(C₁-C₁₀)alkyl.

In another embodiment, (C═O)_(r)O_(s)(C₁-C₁₀)alkyl is methyl.

In another embodiment, (C₀-C₆)alkyl-NR^(a)R^(b) is

or a pharmaceutically acceptable salt thereof.

Compounds according to Figure I may be prepared by various processesknown to one of skill in the art. In particular, compounds according toFigure I, wherein A is CH, B is N, C is CH and R¹ is H may be preparedby the following process:

a) a compound according to Figure IV

Figure IV

wherein Y and Z constitute (C₁-C₆)alkyl groups and R² and R³ are asabove-defined is reacted with a compound according to Figure VFigure V

wherein R⁴ and R⁵ are as above-defined.

Compounds according to Figure II may be prepared by various processesknown to one of skill in the art. In particular, compounds according toFigure II, wherein A is N, B is N, C is CH and R¹ is H may be preparedby the following process:

a) a compound according to Figure VI

Figure VI

wherein Y and Z constitute (C₁-C₆)alkyl groups and R² and R³ are asabove-defined is reacted with a compound according to Figure VIIFigure VII

wherein D and R⁴ are as above-defined.

Compounds according to Figure III may be prepared by various processesknown to one of skill in the art. In particular, compounds according toFigure III, wherein X is O may be prepared by the following process:

a) a compound according to Figure VIII

Figure VIII

wherein R¹ and R² are as above-defined is reacted under basic conditionswith a compound according to Figure IXFigure IX

wherein X is a (C₁-C₆)alkyl group and A is as above-defined.

In one embodiment, the compounds utilized by the methods of theinvention are none of those disclosed in U.S. Pat. No. 5,385,915(incorporated herein by reference in its entirety), particularly atcolumn 11, lines 13-20.

Candidate compounds may be routinely assayed to determine which areappropriate ATP modulators using standard assays, e.g., Xu et al., 1997,Proc. Natl. Acad. Sci. USA 94: 3748-3752 or Zhang et al., 2001,Biochemistry 40: 5049-5055.

Preferably the compound (or agent) administered in the method oftreatment can cross through the blood brain barrier in sufficientquantities and at a sufficient rate so as to allow the treatment of anAβ-related disorder. In one such embodiment, the compound isadministered intravenously. In another embodiment, the compound isadministered orally. More preferably the compound can cross the bloodbrain barrier without a carrier (for methods and routes ofadministration, see Section 5.1.2.5).

Aβ, however, exists not only in brain tissues and in the central nervoussystem (CNS), but also outside the brain in peripheral tissues, such asblood and in most organs where Aβ is also produced by the processing ofAPP. A balance exists between brain Aβ and Aβ outside the brain(DeMattos et al, 2001, Proc. Natl. Acad. Sci. USA 98(15):8850-8855).Reduction of Aβ in the periphery results in a reduction of brain Aβ,presumably by re-partitioning of soluble Aβ. Thus, even compounds thatdo not cross the blood-brain barrier can be utilized in modulating Aβlevels and preventing or treating an Aβ-related disorder, or a symptomthereof.

5.1.2.2. Pharmaceutical Compositions and Formulations

The present invention provides pharmaceutical compositions of theagents, drugs or compounds of the invention disclosed hereinabove.

In one embodiment, the invention provides a pharmaceutical compositioncomprising an amount of a compound described hereinabove sufficient tomodulate Aβ levels in a cell or tissue, and a pharmaceuticallyacceptable excipient or carrier.

In another embodiment, the pharmaceutical composition comprising acompound that lowers ATP-dependent Aβ levels in a cell or tissue, acompound selected from the group consisting of: an NSAID, e.g. sulindacsulfide, flufenamic acid, ibuprofen, flurbiprofen, fenoprofen, mefenamicacid, indomethacin, carprofen, meclofenamic acid, an antioxidant, e.g.,vitamin E, vitamin C, curcumin, and Gingko biloba, a non-selective COXinhibitor and an acetylcholinesterase inhibitor, and a pharmaceuticallyacceptable excipient or carrier.

The agent, drug or compound, or their physiologically acceptable saltsor solvates, may be formulated for administration for injection, or fororal, topical, nasal, inhalation, insufflation (either through the mouthor the nose) buccal, parenteral, rectal administration or other forms ofadministration. In certain embodiments, administration is accomplishedby intrathecal administration via osmotic pumps, by oral gavage, or byintraperiotoneal injection.

The invention provides pharmaceutical compositions comprising sufficientamounts of an agent(s) of the invention together with pharmaceuticallyacceptable diluents, preservatives, solubilizers, emulsifiers,adjuvants, excipients and/or carriers. Such compositions includediluents of various buffer content (e.g., Tris-HCl, acetate, phosphate),pH and ionic strength; additives such as detergents and solubilizingagents (e.g., Tween 80, Polysorbate 80), anti-oxidants (e.g., ascorbicacid, sodium metabisulfite), preservatives (e.g., Thimerosal, benzylalcohol) and bulking substances (e.g., lactose, mannitol).

The compositions may also be incorporated into particulate preparationsof polymeric compounds such as polylactic acid, polyglycolic acid, etc.or liposomes. Hyaluronic acid may also be used. Biocompatible absorbablepolymers may be selected from the group consisting of aliphaticpolyesters, copolymers and blends, which include, but are not limitedto, homopolymers and copolymers of lactide (which include D-, L-, lacticacid and D-, L- and meso lactide), glycolide (including glycolic acid),epsilon-caprolactone, p-dioxanone (1,4-dioxan-2-one, which is disclosedin U.S. Pat. No. 4,052,988), alkyl substituted derivatives ofp-dioxanone (i.e., 6,6-dimethyl-1,4-dioxan-2-one which is disclosed inU.S. Pat. No. 5,703,200), triethylene carbonate (1,3-dioxan-2-one),alkyl substituted derivatives of 1,3-dioxanone (which are disclosed inU.S. Pat. No. 5,412,068), delta-valerolactone, beta-butyrolactone,gamma-butyrolactone, epsilon-decalactone, hydroxybutyrate,hydroxyvalerate, 1,4-dioxepan-2-one (disclosed in U.S. Pat. No.4,052,988 and its dimer 1,5,8,12-tetraoxacyclotetradecane-7,14 dione),1,5-dioxepan-2-one, and polymer blends thereof.

Such compositions may influence physical state, stability, rate of invivo release, and rate of in vivo clearance of the present proteins andderivatives. See, e.g., Remington's Pharmaceutical Sciences, 18th ed.,(1990, Mack Publishing Co., Easton, Pa. 18042) pages 1435-1712). Thecompositions may be prepared in liquid form, or be in dried powder, suchas lyophilized form.

Contemplated for use herein are oral solid dosage forms, which aredisclosed generally in Remington=s Pharmaceutical Sciences, 18th Ed.1990 (Mack Publishing Co. Easton Pa. 18042) at Chapter 89. Solid dosageforms include tablets, capsules, pills, troches or lozenges, cachets orpellets. Also, liposomal or proteinoid encapsulation may be used toformulate the present compositions (as, for example, proteinoidmicrospheres reported U.S. Pat. No. 4,925,673). Liposomal encapsulationmay be used and the liposomes may be derivatized with various polymers(e.g., U.S. Pat. No. 5,013,556). A description of possible solid dosageforms for the therapeutic is given by Marshall, K. In: ModernPharmaceutics Edited by G. S. Banker and C. T. Rhodes Chapter 10, 1979).In general, the formulation will include the agent and inert ingredients(which allow for protection against the stomach environment and releaseof the biologically active material in the intestine).

To ensure full gastric resistance a coating impermeable to at least pH5.0 is useful. Examples of the more common inert ingredients that areused as enteric coatings are cellulose acetate trimellitate (CAT),hydroxypropylmethylcellulose phthalate (HPMCP), HPMCP 50, HPMCP 55,polyvinyl acetate phthalate (PVAP), Eudragit L3OD, Aquateric, celluloseacetate phthalate (CAP), Eudragit L, Eudragit S, and Shellac. Thesecoatings may be used as mixed films.

A coating or mixture of coatings can also be used on tablets, which arenot intended for protection against the stomach. This can include sugarcoatings, or coatings that make the tablet easier to swallow. Capsulesmay consist of a hard shell (such as gelatin) for delivery of drytherapeutic, i.e., powder; for liquid forms, a soft gelatin shell may beused. The shell material of cachets may be thick starch or other ediblepaper. For pills, lozenges, molded tablets or tablet triturates, moistmassing techniques can be used.

The therapeutic can be included in the formulation as finemulti-particulates in the form of granules or pellets. The formulationof the material for capsule administration can also be as a powder,lightly compressed plugs or even as tablets. The therapeutic can also beprepared by compression.

Colorants and flavoring agents may all be included. For example, theprotein (or derivative) may be formulated (such as by liposome ormicrosphere encapsulation) and then further contained within an edibleproduct, such as a refrigerated beverage containing colorants andflavoring agents.

One may dilute or increase the volume of the therapeutic with an inertmaterial or filler. These diluents or fillers can include carbohydrates,especially mannitol, a-lactose, anhydrous lactose, cellulose (e.g.,microcrystalline cellulose), sucrose, calcium hydrogen phosphatemodified dextrans and starch. Certain inorganic salts may be also beused as fillers including calcium triphosphate, magnesium carbonate andsodium chloride. Some commercially available diluents are Fast-Flo,Emdex, STA-Rx 1500, Emcompress and Avicell.

Disintegrants may be included in the formulation of the therapeutic intoa solid dosage form. Materials used as disintegrates include, but arenot limited to, starch (e.g., potato starch or the commercialdisintegrant based on starch, Explotab). Sodium starch glycolate,Amberlite, sodium carboxymethylcellulose, ultramylopectin, sodiumalginate, gelatin, orange peel, acid carboxymethyl cellulose, naturalsponge and bentonite may all be used. Another form of the disintegrantsare the insoluble cationic exchange resins. Powdered gums may be used asdisintegrants and as binders and these can include powdered gums such asagar, Karaya or tragacanth. Alginic acid and its sodium salt are alsouseful as disintegrants.

Binders may be used to hold the therapeutic agent together to form ahard tablet and include materials from natural products such as acacia,tragacanth, starch (e.g., pregelatinised maize starch) and gelatin.Others include methyl cellulose (MC), ethyl cellulose (EC) andcarboxymethyl cellulose (CMC). Polyvinyl pyrrolidone (PVP) andhydroxypropylmethyl cellulose (HPMC) can both be used in alcoholicsolutions to granulate the therapeutic.

An anti-frictional agent may be included in the formulation of thetherapeutic to prevent sticking during the formulation process.Lubricants may be used as a layer between the therapeutic and the diewall, and these can include but are not limited to; stearic acidincluding its magnesium and calcium salts, polytetrafluoroethylene(PTFE), liquid paraffin, vegetable oils and waxes, talc and silica.Soluble lubricants may also be used such as sodium lauryl sulfate,magnesium lauryl sulfate, polyethylene glycol of various molecularweights, Carbowax 4000 and 6000.

Glidants that can improve the flow properties of the drug duringformulation and to aid rearrangement during compression can be added.The glidants can include starch, talc, pyrogenic silica and hydratedsilicoaluminate.

To aid dissolution of the therapeutic into the aqueous environment asurfactant can be added as a wetting agent. Surfactants may includeanionic detergents such as sodium lauryl sulfate, dioctyl sodiumsulfosuccinate and dioctyl sodium sulfonate. Cationic detergents can beused and can include benzalkonium chloride or benzethomium chloride. Thelist of potential non-ionic detergents that can be included in theformulation as surfactants are lauromacrogol 400, polyoxyl 40 stearate,polyoxyethylene hydrogenated castor oil 10, 50 and 60, glycerolmonostearate, polysorbate 40, 60, 65 and 80, sucrose fatty acid ester,methyl cellulose and carboxymethyl cellulose. These surfactants can bepresent in the formulation of the protein or derivative either alone oras a mixture in different ratios.

Additives that potentially enhance uptake of the agent are, for example,the fatty acids oleic acid, linoleic acid and linolenic acid.

Controlled release oral formulation may be desirable. The agent can beincorporated into an inert matrix that permits release by eitherdiffusion or leaching mechanisms, e.g., gums. Slowly degeneratingmatrices may also be incorporated into the formulation. Some entericcoatings also have a delayed release effect.

Another form of a controlled release of this therapeutic is by a methodbased on the Oros therapeutic system (Alza Corp.), i.e. the drug isenclosed in a semipermeable membrane, which allows water to enter and topush the drug out through a single small opening due to osmotic effects.

Other coatings may be used for the formulation. These include a varietyof sugars that can be applied in a coating pan. The therapeutic agentcan also be given in a film coated tablet and the materials used in thisinstance are divided into two groups. The first are the nonentericmaterials and include methyl cellulose, ethyl cellulose, hydroxyethylcellulose, methylhydroxy-ethyl cellulose, hydroxypropyl cellulose,hydroxypropyl-methyl cellulose, sodium carboxy-methyl cellulose,providone and the polyethylene glycols. The second group consists of theenteric materials that are commonly esters of phthalic acid.

A mix of materials can be used to provide the optimum film coating. Filmcoating may be carried out in a pan-coater or in a fluidized bed or bycompression coating.

Liquid preparations for oral administration may take the form of, forexample, solutions, syrups or suspensions, or they may be presented as adry product for constitution with water or other suitable vehicle beforeuse. Such liquid preparations may be prepared by conventional means withpharmaceutically acceptable additives such as suspending agents (e.g.,sorbitol syrup, cellulose derivatives or hydrogenated edible fats);emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles(e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetableoils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates orsorbic acid). The preparations may also contain buffer salts, flavoring,coloring and sweetening agents as appropriate.

Nasal delivery of the agent is also contemplated. Nasal delivery allowsthe passage of the protein to the blood stream directly afteradministering the therapeutic product to the nose, without the necessityfor deposition of the product in the lung. Formulations for nasaldelivery include those with dextran or cyclodextran.

For administration by inhalation, the compounds for use according to thepresent invention are conveniently delivered in the form of an aerosolspray presentation from pressurized packs or a nebulizer, with the useof a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol the dosage unitmay be determined by providing a valve to deliver a metered amount.Capsules and cartridges of e.g., gelatin for use in an inhaler orinsufflator may be formulated containing a powder mix of the compoundand a suitable powder base such as lactose or starch.

The compounds may be formulated for parenteral administration byinjection, e.g., by bolus injection or continuous infusion. Formulationsfor injection may be presented in unit dosage form, e.g., in ampoules orin multi-dose containers, with an added preservative. The compositionsmay take such forms as suspensions, solutions or emulsions in oily oraqueous vehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the activeingredient may be in powder form for constitution with a suitablevehicle, e.g., sterile pyrogen-free water, before use.

The compounds may also be formulated in rectal compositions such assuppositories or retention enemas, e.g., containing conventionalsuppository bases such as cocoa butter or other glycerides.

In addition to the formulations disclosed previously, the compounds mayalso be formulated as a depot preparation. Such long acting formulationsmay be administered by implantation (for example subcutaneously orintramuscularly) or by intramuscular injection. Thus, for example, thecompounds may be formulated with suitable polymeric or hydrophobicmaterials (for example as an emulsion in an acceptable oil) or ionexchange resins, or as sparingly soluble derivatives, for example, as asparingly soluble salt.

The compositions may, if desired, be presented in a pack or dispenserdevice that may contain one or more unit dosage forms containing theactive ingredient. The pack may for example comprise metal or plasticfoil, such as a blister pack. The pack or dispenser device may beaccompanied by instructions for administration.

Other pharmaceutical compositions can be co-administered (sequentiallyor concurrently) with the pharmaceutical composition of the invention.Co-administration can be accomplished by administration of such otherpharmaceutical compositions in the same or in a different pharmaceuticalcomposition as those described above. In one embodiment, an NSAID isco-administered, e.g., sulindac sulfide, flufenamic acid, ibuprofen,flurbiprofen, fenoprofen, mefenamic acid, indomethacin, carprofen ormeclofenamic acid. In another embodiment, a non-selective COX inhibitoris co-administered. In another embodiment, a non-specific secretaseinhibitor is co-administered. In another embodiment, an antioxidant isco-administered. In specific embodiments, the antioxidant is selectedfrom the group consisting of vitamin E, vitamin C, curcumin, and Gingkobiloba. In another embodiment, an acetylcholinesterase inhibitor isco-administered.

5.1.2.3. Articles of Manufacture

The present invention also provides an article of manufacture comprisingpackaging material and a pharmaceutical composition of the inventioncontained within the packaging material, said pharmaceutical compositionin a form suitable for administration to a subject, preferably a human,or in a format that can be diluted or reconstituted for administrationto the subject. In one embodiment, the article of manufacture furthercomprises printed instructions and/or a label directing the use oradministration of the pharmaceutical composition.

In particular, the article of manufacture can comprise packagingmaterial and a pharmaceutical composition comprising a compound thatlowers ATP-dependent Aβ levels, and a pharmaceutically acceptablecarrier contained within the packaging material, said pharmaceuticalcomposition in a form suitable for administration to a subject. Such anarticle of manufacture can further comprise printed instructionsregarding the use or administration of the pharmaceutical composition.For example, the instructions suggest a dosing regimen for theprevention, treatment, or amelioration of a symptom of an Aβ-relateddisorder such as for the prevention, treatment, or amelioration of asymptom of Alzheimer's disease. An article of manufacture of theinvention can also further comprise a label regarding the use oradministration of the pharmaceutical composition. For example, the labelcan suggest a dosing regimen for the prevention, treatment, oramelioration of a symptom of an Aβ-related disorder such as Alzheimer'sdisease. In one embodiment, the article of manufacture can furthercomprises an antioxidant, a non-selective COX inhibitor or anacetylcholinesterase inhibitor.

As with any pharmaceutical product, the packaging material and containerof the articles of manufacture of the invention are designed to protectthe stability of the product during storage and shipment. Morespecifically, the invention provides an article of manufacturecomprising packaging material, such as a box, bottle, tube, vial,container, sprayer, insufflator, intravenous (i.v.) bag, envelope andthe like; and at least one unit dosage form of a pharmaceuticalcomposition of the invention contained within said packaging material.

5.1.2.4. Dosages

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD50 (the dose lethal to 50% of thepopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD50/ED50.Compounds that exhibit large therapeutic indices are preferred. Whilecompounds that exhibit toxic side effects may be used, care should betaken to design a delivery system that targets such compounds to thesite of affected tissue in order to minimize potential damage touninfected cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED50 with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the invention, the therapeutically sufficient dose canbe estimated initially from cell culture assays. A dose may beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC50 (i.e., the concentration ofthe test compound that achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma may bemeasured, for example, by high performance liquid chromatography.

In certain embodiments, treatment can begin at the first signs of mildcognitive impairment in a subject or patient, using standard methods forassessing mild cognitive impairment known in the art. In otherembodiments, for example when a subject is at risk for familialAlzheimer's disease, treatment can begin before onset of mild cognitiveimpairment.

In certain embodiments, a treatment regimen can be used as aprophylactic or preventative measure. For example, in certainembodiments, a treatment regimen is administered to an elderly person,e.g., age 65 or older. In another embodiment, demographic data availablein the art may be consulted to determine the average age of onset ofAlzheimer's disease in a population of interest.

In a specific embodiment, a pharmaceutical composition of the inventionis administered chronically, e.g., daily. In one embodiment, apharmaceutical composition, e.g., a blood-brain permeable version of themesylate salt of STI-571 (or its mesylate salt) is administered dailyvia oral administration, with a minimum of 10 μg/day and a maximum of800 μg/day administered.

5.1.2.5. Routes of Administration

The component or components of a therapeutic composition of theinvention may be introduced parenterally, topically, or transmucosally,e.g., orally, nasally, or rectally, or transdermally. In certainembodiments, administration is parenteral, e.g., via intravenousinjection, and also including, but not limited to, intra-arteriole,intramuscular, intradermal, subcutaneous, intraperitoneal,intraventricular, and intracranial administration. In certainembodiments, the component or components of a therapeutic composition ofthe invention is introduced by intrathecal administration via osmoticpumps (e.g., a refillable osmotic pump), by oral gavage, or byintraperiotoneal injection.

In preferred embodiments of the invention, an agent (or drug orcompound) can cross and more preferably, readily pass through theblood-brain barrier, which permits, e.g., oral, parenteral orintravenous administration. Alternatively, the agent can be modified orotherwise altered so that it can cross or be transported across theblood brain barrier. Many strategies known in the art are available formolecules crossing the blood-brain barrier, including but not limitedto, increasing the hydrophobic nature of a molecule; introducing themolecule as a conjugate to a carrier, such as transferring, targeted toa receptor in the blood-brain barrier, or to docosahexaenoic acid etc.

In a specific embodiment, the agent is delivered orally and can readilypass through the blood-brain barrier.

In another embodiment, an agent of the present invention is administeredvia the standard procedure of drilling a small hole in the skull toadminister the agent.

In other embodiments, the molecule can be administered intracranially orintraventricularly. In another embodiment, osmotic disruption of theblood-brain barrier can be used to effect delivery of agent to the brain(Nilayer et al., 1995, Proc. Natl. Acad. Sci. USA 92:9829-9833). In yetanother embodiment, an agent can be administered in a liposome targetedto the blood-brain barrier. Administration of pharmaceutical agents inliposomes is known (see Langer, 1990, Science 249:1527-1533; Treat etal., 1989, in Liposomes in the Therapy of infectious Disease and Cancer,Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 317-327 and353-365). All of such methods are envisioned in the present invention.

The rate and extent of entry of a compound into the brain are generallyconsidered to be determined by partition coefficient, ionizationconstant(s), and molecular size. The octanol water system, for example,has received particular attention, and Hansch and coworkers havesuggested that a partition coefficient in this system of about 100 isoptimal for entry into the central nervous system (CNS) (Giave andHansch, 1972, J. Pharm. Sci. 61:589; Hansch et al., 1987, J. Pharm. Sci.76:663). The octanol-water partition system provides a qualitativeindication of the capability of a compound to cross the blood-brainbarrier. Other factors, besides the octanol-water partition can bemanipulated to influence the propensity to cross the blood-brainbarrier. For example, decreasing over-all hydrogen binding ability of acompound can be used to facilitate a compound's crossing of theblood-brain barrier. Further, methodology as used by Begley et al. canbe used and includes: (1) measuring the brain uptake index (BUI) withthe equation for a tritiated agent compound:BUI=[(brain ³H/brain ¹⁴C)/(injectate ³H/injectate ¹⁴C)]×100where the ¹⁴C reference compound is ¹⁴C butanol or an analogous solvent;(2) Brain perfusion studies; (3) Intravenous bolus injection studies;and (4) Studies with cultured cerebral capillary endothelium.

In another embodiment used to reduce systemic side effects, thetherapeutic compound is delivered in a vesicle, in particular a liposome(see Langer, 1990, Science 249:1527-1533; Treat et al., 1989, inLiposomes in the Therapy of Infectious Disease and Cancer,Lopez-Berestein and Fidler (eds.), Liss: New York, pp. 317-327 and353-365).

In another embodiment, the therapeutic compound can be delivered in acontrolled release system. For example, the agent may be administeredusing intravenous infusion, an implantable osmotic pump, a transdermalpatch, liposomes, or other modes of administration. In one embodiment, apump may be used (see Langer, 1990, Science 249:1527-1533; Sefton, 1987,CRC Crit. Ref. Biomed. Eng. 14:201; Buchwald et al., 1980, Surgery88:507; Saudek et al., 1989, N. Engl. J. Med. 321:574). In anotherembodiment, polymeric materials can be used (see Medical Applications ofControlled Release, Langer and Wise (eds.), 1974, CR^(c) Press: BocaRaton, Fla.; Controlled Drug Bioavailability, Drug Product Design andPerformance, Smolen and Ball (eds.), 1984, Wiley: New York; Ranger andPeppas, 1983, J. Macromol. Sci. Rev. Macromol. Chem. 23:61; see alsoLevy et al., 1985, Science 228:190; During et al., 1989, Ann. Neurol.25:351; Howard et al., 1989, J. Neurosurg. 71:105). In yet anotherembodiment, a controlled release system can be placed in proximity ofthe therapeutic target, i.e., the brain, thus requiring only a fractionof the systemic dose (see, e.g., Goodson, 1984, in Medical Applicationsof Controlled Release, supra, vol. 2, pp. 115-138). Other controlledrelease systems are discussed in the review by Langer (1990, Science249:1527-1533).

5.2. Methods for Screening for Compounds that Modulate Aβ Levels

The present invention provides, in vivo, in situ, and in vitro, methodsof identifying an agent, drug or compound for modulating Aβ levelsexhibited by a cell or tissue. In certain embodiments, the agent has theability to either stimulate or inhibit Aβ levels through modulation ofthe activity of γ-secretase. In one embodiment, the γ-secretase activityis ATP-dependent γ-secretase activity.

In one aspect, the method comprises identifying an agent to be testedfor an ability to treat a disorder, including, but not limited to, anAβ-related disorder. Such methods can be used alone or in conjunctionwith each other.

In certain embodiments, the invention provides methods of identifying anagent, drug or compound that modulate ATP-dependent Aβ peptideproduction and/or levels of Aβ in cells and tissues.

In certain embodiments, the invention provides methods of identifying anagent, drug or compound for modulating γ-secretase activity based on thedetermination of γ-secretase activity both before and after treatment ofan ATP-dependent Aβ generating system with the compound to be tested.Such a system can be established from either broken cells (Xu et al.,1997, Proc. Natl. Acad. Sci. USA 94: 3748-3752) or isolated membranes(Zhang et al., 2001, Biochemistry 40: 5049-5055).

The invention also provides method of identifying a compound thatmodulates Aβ levels exhibited by a cell or tissue comprising:

-   -   (a) determining a first level of γ-secretase activity in said        cell or tissue;    -   (b) contacting said cell or tissue with a test compound; and    -   (c) determining a second level of γ-secretase activity in said        cell or tissue,        wherein a difference in said first level and said second level        of γ-secretase activity is indicative of the ability of said        test compound to modulate Aβ levels.

In one embodiment, the difference in γ-secretase activity is indicativeof the ability of said test compound to modulate the Aβ levels. Inanother embodiment, Aβ levels are modulated. In another embodiment, theγ-secretase activity is an ATP-dependent enzymatic activity. In anotherembodiment, the γ-secretase activity is production of Aβ.

The invention also provides a method of identifying a compound thatmodulates Aβ levels exhibited by a cell or tissue comprising:

-   -   (a) contacting said cell or tissue with a test compound; and    -   (b) determining a level of activity of γ-secretase in said cell        or tissue;        wherein a difference in said level and a control level of        γ-secretase activity in a comparable cell or tissue not        contacted with the test compound is indicative of the ability of        said test compound to modulate Aβ levels.

In one embodiment, the difference in γ-secretase activity is indicativeof the ability of said test compound to modulate the AD levels. Inanother embodiment, Aβ levels are modulated. In another embodiment, theγ-secretase activity is an ATP-dependent enzymatic activity. In anotherembodiment, the γ-secretase activity is production of Aβ.

The invention also provides a method for identifying an agent to betested for an ability to treat an Aβ-related disorder in a patient inneed of such treatment comprising:

-   -   (a) contacting in a cell or tissue γ-secretase with a potential        agent; and    -   (b) detecting the amount of γ-secretase activity        wherein the agent is identified if a decrease in γ-secretase        activity is detected in the presence of the potential agent and        wherein the agent modulates Aβ levels.

In one embodiment, the ability to treat the Aβ-related disorder istested. In another embodiment, the γ-secretase activity is anATP-dependent enzymatic activity. In another embodiment, the γ-secretaseactivity is production of Aβ.

In another aspect, the invention provides a method for identifying anagent to be tested for an ability to modulate Aβ levels exhibited by acell or tissue comprising:

-   -   (a) determining a first level of γ-secretase activity in said        cell or tissue;    -   (b) contacting said cell or tissue with a potential agent; and    -   (c) determining a second level of γ-secretase activity in said        cell or tissue,        wherein a difference in said first level and said second level        of γ-secretase activity is indicative of the ability of said        potential agent to modulate Aβ levels. In one embodiment, the        method comprises the additional step of:    -   (d) determining whether the Aβ levels are modulated.

In another embodiment, the γ-secretase activity is an ATP-dependentenzymatic activity. In another embodiment, the γ-secretase activity isproduction of Aβ.

The invention also provides a method for identifying an agent to betested for an ability to modulate Aβ levels exhibited by a cell ortissue comprising:

-   -   (a) contacting said cell or tissue with a potential agent; and    -   (b) determining a level of γ-secretase activity in said cell or        tissue;        wherein a difference in said level and a control level of        γ-secretase activity in a comparable cell or tissue not        contacted with the test compound is indicative of the ability of        said potential agent to modulate Aβ levels.

In one embodiment, the method comprises the additional step of:

-   -   (c) determining whether the Aβ levels are modulated.

In another embodiment, the γ-secretase activity is an ATP-dependentenzymatic activity.

In another embodiment, the γ-secretase activity is production of Aβ.

The invention also provides a method for selecting a potentialtherapeutic agent for use in the treatment of an Aβ-related disordercomprising:

-   -   (a) administering a potential therapeutic agent to an animal;    -   (b) measuring the response of said animal to said potential        therapeutic agent;    -   (c) comparing the response of said animal with that of a control        animal to which the potential therapeutic agent has not been        administered; and    -   (d) selecting a potential therapeutic agent based on the        difference in responses observed between said animal and said        control animal,        wherein the potential therapeutic agent modulates ATP-dependent        γ-secretase activity.

In one embodiment, the animal is a guinea pig. In another embodiment,the disorder is Alzheimer's disease.

The invention also provides a method for selecting a potentialtherapeutic agent for use in the treatment of an Aβ-related disordercomprising:

-   -   (a) administering a potential therapeutic agent to an animal;    -   (b) measuring the response of said animal to administration of        an agent that modulates activity of γ-secretase;    -   (c) comparing the response of said animal with that of a control        animal to which the potential therapeutic agent has not been        administered; and    -   (d) selecting a potential therapeutic agent based on the        difference in responses observed between said animal and said        control animal,        wherein the potential therapeutic agent modulates ATP-dependent        γ-secretase activity.

In one embodiment, the animal is a guinea pig. In another embodiment,the disorder is Alzheimer's disease.

The invention also provides a method for selecting a potentialtherapeutic agent for use in the treatment of an Aβ-related disordercomprising:

-   -   (a) administering a potential therapeutic agent to an animal;    -   (b) measuring the response of said animal, wherein the response        is selected from the group consisting of:        -   (i) exhibition of behavior in a Morris water maze; and        -   (i) exhibition of behavior in a Y-maze    -   (c) comparing the response of said animal with that of a control        animal to which the potential therapeutic agent has not been        administered; and    -   (d) selecting a potential therapeutic agent based on the        difference in responses observed between said animal and said        control animal,        wherein the potential therapeutic agent modulates Aβ levels.

In one embodiment, the animal is a guinea pig. In another embodiment,the disorder is Alzheimer's disease.

The invention also provides a method for preventing, delaying orreversing the progression of an Aβ-related disorder comprising:

-   -   (a) identifying a mammal in need of prevention, delay, or        reversal of the progression of the disorder; and    -   (b) administering to said mammal an amount of an agent        sufficient to modulate ATP-dependent γ-secretase activity,        wherein Aβ levels are modulated.

In one embodiment, the agent inhibits or decreases ATP-dependentγ-secretase activity.

In another embodiment, the mammal is human. In another embodiment, thedisorder is Alzheimer's disease.

In another embodiment, the agent promotes or increases ATP-dependentγ-secretase activity.

In another embodiment, the agent is administered orally.

In another embodiment, the agent is administered with a NSAID. Inspecific embodiment, the NSAID is sulindac sulfide, flufenamic acid,ibuprofen, flurbiprofen, fenoprofen, mefenamic acid, indomethacin,carprofen, meclofenamic acid.

In another embodiment, the agent is administered with an antioxidant. Inanother embodiment, the antioxidant is selected from the groupconsisting of vitamin E, vitamin C, curcumin, and Gingko biloba.

In another embodiment, the agent the agent is administered with anacetylcholinesterase inhibitor.

According to the invention, a “control” level means a separate baselinelevel exhibited by a comparable cell or tissue not contacted with a testcompound or a level that is exhibited by a cell or tissue prior tocontacting it with a test compound. A control level can return to aknown standard reference level.

As would be clearly understood by a person of ordinary skill in the art,any and/or all of the embodiments disclosed herein for identifying anagent, drug or compound that can modulate Aβ levels, including suchprocedures that incorporate rational drug design, as disclosed herein,can be combined to form additional drug screens and assays, all of whichare contemplated by the present invention.

According to the methods of the invention, γ-secretase activity and/orAβ level may be determined both before and after treatment of cells ortissues with a test compound.

One of skill would understand that according to the invention, once acompound is identified as capable of producing, e.g., γ-secretaseactivity and/or Aβ level similar to those produced by known ameliorativecompounds, the compound may be used to treat an Aβ-related disorder.Such conditions would include, but not be limited to, Alzheimer'sdisease, Down's syndrome and inclusion body myositis. In the context ofthe present invention, the compounds identified would be administered asa sufficient dose or amount which can be determined by one of skill inthe art based on data from studies such as presented in thisspecification. Such data would include, but not be limited to, resultsfrom IC50 determinations.

To determine whether a modulator is a selective ATP modulator, theskilled artisan would determine, using methods well known in the art,whether a candidate modulator discriminates among the molecules thataffect ATP function in a cell of interest, and whether it only modulatesa specific individual molecule, or class or subset of the molecules. Forexample, a compound may be assayed for activity in inhibiting acollection of various kinases, and would be recognized as a selectiveATP modulator if it modulates ATP function in a related class ofkinases. In certain embodiments, a battery of standard kinase assays isrun to determine whether a modulator only modulates ATP function of aspecific subset or class of kinases. In a specific embodiment, astandard luciferase (ATP-dependency) assay can be used. In certainembodiments, a non-specific ATP modulator will modulate luciferaseproduction in the assay, whereas a specific inhibitor will not.

In other embodiments, a selective ATP modulator does not affect tauphosphorylation (see, e.g., U.S. Pat. No. 5,955,444) or Notch cleavage(see Section 10).

In one embodiment, the selectivity profile of the ATP modulator issimilar or identical to that of STI-571 (or its mesylate salt,GLEEVEC™), STI-571 variant WGB-BC-15 or any of Compounds 1-5.

In certain embodiments, the modulator modulates an ATP-dependentcellular process, as disclosed herein. In other embodiments, however, itis contemplated that the modulator may modulate other energy-dependent(e.g., GTP-dependent) processes that utilize sources of energy otherthan ATP (e.g., GTP).

The present invention also provides in vivo methods of identifyingagents that can modulate the level of Aβ via modulation of γ-secretaseactivity. Such methods can be employed alone or in conjunction with invitro and in situ methods as disclosed herein.

According to the invention, in one embodiment of the invention, theskilled artisan would administer a modulator of Aβ levels, e.g., an ATPmodulator or an inhibitor of γ-secretase activity, based on results ofscreening for levels of Aβ peptides produced in the presence and absenceof the inhibitor. Such screening of compounds is routine to one of skillin the art (Durkin et al., 1999, J. Biol. Chem. 274 (29): 20499-20504;Petanceska and Gandy, 1999, J. Neurochem. 73: 2316-2320; De Strooper etal., 1999, Nature 398: 518-522; Vandermeerena et al., 2001, NeuroscienceLetters 315: 145-148). Any compound identified as having selectiveγ-secretase inhibitory activity or to selectively inhibit Aβ productionwould be administered, e.g., in a pharmaceutically acceptable vehicle,to an animal, e.g., a human.

One such in vivo method comprises administering the agent to a non-humanmammal. The amount (and/or rate) of activation of γ-secretase is thendetermined. An agent is identified as capable of modulating the level ofAβ, via modulation of γ-secretase activity, when the amount (and/orrate) of γ-secretase activation is increased or decreased in thepresence of the agent relative to in the absence of the agent. In aspecific embodiment, the γ-secretase activity is ATP-dependentγ-secretase activity. In one embodiment, the non-human mammal is arodent, e.g., a wild-type guinea pig. In other embodiments, thenon-human mammal is an animal model for a disease or disorder. Suchanimal models are disclosed herein.

In another embodiment, an experimental animal is used to ascertain theeffect of a potential agent on a disorder, including but not limited toan Aβ-related disorder. A potential modulator that ameliorates thedisorder can then be selected.

For example, in certain embodiments, a learning and/or memory behavioralresponse of an animal can be determined in the presence and absence ofthe agent. In specific embodiment, learning and/or memory behavioralresponse of the animal, e.g., a transgenic mouse such as a Tg2576 mouse.The Tg2576 transgenic mouse expresses human APP 695, develops theneuropathological signs of Alzheimer's disease, including amyloidplaques, and exhibits learning and memory deficits (Hsiao et al., 1996,Science 274, 99-102; U.S. Pat. No. 5,877,399). Another useful model is atransgenic mouse expressing human APP and a familial Alzheimer's diseasegene (“human APP-Swedish”) (Borchelt et al., 1996, Neuron 17:1005-1013).The learning and/or memory behavioral response of such an animal modelcan be measured using standard methods, e.g., a Y-maze or a Morris watermaze (see, e.g., U.S. Pat. No. 5,877,399).

Methods of testing a potential therapeutic agent (e.g., a candidatedrug, potential modulator, etc.) in animals or animal models are wellknown in the art. Thus potential therapeutic agents can be used to treatwhole animals. The potential modulators can be administered by a varietyof ways including topically, orally, subcutaneously, orintraperitoneally (such as by intraperitoneal injection) depending onthe proposed use. Optimal dose will be empirically defined.

The potential efficacy of these compounds in relieving pathologicalsymptoms of a disorder, including but not limited to, an Aβ-relateddisorder, can be assessed in animal models for disease. For example, inone embodiment, the animal model is a Tg2576, a transgenic mouse thatexpresses human APP 695. The Tg2576 mouse develops the neuropathologicalsigns of Alzheimer's disease, including amyloid plaques, and exhibitslearning and memory deficits (Hsiao et al., 1996, Science 274, 99-102;U.S. Pat. No. 5,877,399). Another useful model is a transgenic mouseexpressing human APP and a familial Alzheimer's disease gene (“humanAPP-Swedish”) (Borchelt et al., 1996, Neuron 17:1005-1013).

In certain embodiments of the invention, modulation of Aβ 1-40 levels isscreened for. Aβ 1-40 is the major product of catabolism of APP byβ-secretase and γ-secretase enzyme activities. The amino acid sequenceof Aβ 1-40 corresponds to residues 597-636 of the 695 amino acid isotypeof human APP:

[SEQ ID NO: 1] Asp Ala Glu Phe Arg His Asp Ser Gly Tyr 1Glu Val His His Gln Lys Leu Val Phe Phe 21Ala Glu Asp Val Gly Ser Asn Lys Gly Ala 31Ile Ile Gly Leu Met Val Gly Gly Val Val.

In other embodiments, modulation of Aβ 1-42 level is screened for. Aβ1-42 is a minor product of APP catabolism, typically produced at 10% thelevel of Aβ 1-40, that aggregates into fibrillar forms very rapidly. Theamino acid sequence of Aβ 1-42 corresponds to residues 597-638 of humanAPP:

[SEQ ID NO: 2] Asp Ala Glu Phe Arg His Asp Ser Gly Tyr 11Glu Val His His Gln Lys Leu Val Phe Phe 21Ala Glu Asp Val Gly Ser Asn Lys Gly Ala 31Ile Ile Gly Leu Met Val Gly Gly Val Val Ile Ala.

Models such as these can be used to assess the efficacy of any potentialtherapeutic agents as disclosed hereinbelow. Generally, at least twogroups of animals are used in the assay, with at least one group being acontrol group in which the administration vehicle is administeredwithout the potential therapeutic agent.

Another aspect of the invention is a method for selecting a therapeuticagent for potential use in the treatment of disorder, including but notlimited to an Aβ-related disorder, which comprises administering asuspected therapeutic agent to an animal model, and measuring and/ordetermining the putative therapeutic agent's effect on any of thephenotypic characteristics of the animal model, which may be believed tobe related to said disorder.

Potential therapeutic agents are selected on the basis of whether thereis a statistical significance between a test response and a normal(i.e., naive or control) response. Potential therapeutic agents areselected that show a statistically significant change in thecharacteristic measured/determined. In a preferred embodiment, theresponse of a control (or vehicle-treated) animal in the presence of atherapeutic agent is characteristically different from the response of acontrol (or vehicle-treated) animal to which the agent has not beenadministered.

A still further aspect of the invention is a method for selecting atherapeutic agent for possible use in the treatment of a disorder,including but not limited to an Aβ-related disorder, which comprisesadministering a suspected therapeutic agent to an animal model for adisorder and measuring and/or determining the putative therapeuticagent's effect on any of the phenotypic characteristics outlined abovewhich may be believed to be related to said disorder.

In other embodiments, the agent is administered along with anantagonist. The amount (and/or rate) of modulation of γ-secretaseactivity is then determined. Since the administration of e.g., anantagonist, in the absence of the agent, should result in an increase inγ-secretase activity, an agent is identified as capable of modulatingthe activity of γ-secretase when the amount (and/or rate) of activationis significantly increased or decreased in the presence of the agentrelative to in the absence of the agent.

In other embodiments, the agent is administered along with a nucleosidetriphosphate. The amount (and/or rate) of modulation of γ-secretaseactivity is then determined. Since the administration of a nucleosidetriphosphate, in the absence of the agent should result in a increase inγ-secretase activity, an agent is identified as capable of modulatingthe activity of γ-secretase when the amount (and/or rate) of activationis significantly increased or decreased in the presence of the agentrelative to in the absence of the agent.

In certain embodiments, combinatorial libraries of chemical compounds,based on different structural skeletons (e.g., purines), as well asunrelated naturally occurring compounds, can be tested as drugcandidates. In a preferred embodiment of this type, the assay isperformed using high throughput technology with automated robotictechnology as disclosed herein. Positive results (“hits”) representeither the reduced or increased activity of γ-secretase, as compared tothe control reactions (in which the drug candidate is not included inthe assay).

Once a drug candidate is selected, structural variants of the drugcandidate can be tested. These compounds can also be scrutinized andmodified with parameters such as membrane permeability, specificity ofeffects, and toxicity. The selected (e.g., the most potent) compounds ofthis secondary screening can then be evaluated in situ and in animalmodels (see Section 9) to determine whether the selected compounds alterthe activity of γ-secretase, and/or induce predicted behavioralalterations with minimal to no side-effects. Such behavioralabnormalities may include, but not be limited to, testing locomotoractivity or learning and memory, as disclosed herein (see also, e.g.,U.S. Pat. No. 5,877,399; and Kosten et al., J. Pharmacol., Exp. Ther.269:137-144 (1994). In specific embodiments, methods for testing forlearning and memory commonly known in the art, e.g., a Y-maze or aMorris water test can be used (see, e.g., U.S. Pat. No. 5,877,399).

These tests can be then be followed by human trials in clinical studies.Alternatively, in certain embodiments, human trials in clinical studiescan be performed without animal testing. Compounds affecting targetsother than γ-secretase can also be similarly screened, using alternativetargets exemplified below.

Alternatively, modulators (e.g., activators or inhibitors) ofγ-secretase activity can be obtained by screening, e.g., a randompeptide library produced by recombinant bacteriophage (see, e.g., Scottand Smith, Science 249:386-390 (1990); Cwirla et al., 1990, Proc. Natl.Acad. Sci. USA 87:6378-6382; and Devlin et al., 1990, Science249:404-406) or a chemical library. Using the “phage method” very largelibraries can be constructed (106-108 chemical entities). A secondapproach may be to use chemical methods, of which the Geysen method(Geysen et al., 1986, Molecular Immunology 23:709-715; Geysen et al.,1987, J. Immunologic Method 102:259-274) and the method of Fodor et al.(1991, Science 251:767-773) are examples. Furka et al. (1988, 14thinternational Congress of Biochemistry, Volume 5, Abstract FR:013;Furka, 1991, Int. J. Peptide Protein Res. 37:487-493), Houghton (U.S.Pat. No. 4,631,211) and Rutter et al. (U.S. Pat. No. 5,010,175) disclosemethods to produce a mixture of peptides. Such peptides can be tested aspotential modulators of γ-secretase activity.

In another aspect, synthetic libraries (Needels et al., 1993, Proc.Natl. Acad. Sci. USA 90:10700-4; Ohlmeyer et al., 1993, Proc. Natl.Acad. Sci. USA 90:10922-10926; Lam et al., International PatentPublication No. WO 92/00252; Kocis et al., International PatentPublication No. WO 94/28028), and the like can be used to screen formodulators of γ-secretase activation, according to the presentinvention. Once a potential modulator is identified, chemical analoguescan be either selected from a library of chemicals as are commerciallyavailable (e.g., from Chembridge Corporation, San Diego, Calif. orEvotec OAI, Abingdon, UK), or alternatively synthesized de novo. Theprospective agent (drug) can be placed into any standard assay to testits effect on the activity of γ-secretase activation. A drug is thenselected that modulates the activity of γ-secretase activation.

The present invention also contemplates screens for small molecules,analogs thereof, as well as screens for natural modulators ofγ-secretase, such as those molecules that bind to and inhibit oractivate, e.g., γ-secretase in vivo. Alternatively, natural productslibraries can be screened using assays of the invention for moleculesthat modulate e.g., γ-secretase activity.

In another aspect of the present invention, a potential modulator can beassayed for its ability to modulate the level of Aβ, eitherindependently, or subsequent to, a binding assay as disclosed herein.Such assays are known in the art. Thus, according to the methods of theinvention, a modulator of γ-secretase activity that modulates the levelof Aβ is then selected

In another embodiment, a potential modulator can be added to a culturedcell line or tissue explant. Samples of the cells or tissues can betreated with various concentrations of a potential modulator and thesamples can then be analyzed for Aβ levels. Potential modulators of Aβlevels can also be tested for example, on intact neurons in situ. Theeffects of these compounds can be tested by empirically defining theoptimal concentration and time of incubation.

The present invention also includes compositions identified by themethods described herein. One of skill in the art would understand thatonce identified as capable of modulating Aβ levels in the method of thepresent invention, the compound of the invention could be usedtherapeutically to modulate Aβ levels in neuronal cells in order totreat conditions in which Aβ levels may be involved.

One of skill will understand that once identified as capable ofmodulating γ-secretase activity in the methods of the present invention,the compound may be used therapeutically to modulate γ-secretaseactivity in cells, e.g., neurons, in order to treat conditions in whichγ-secretase activity may be involved. Such conditions include, but arenot limited to, an Aβ-related disorder.

The present invention further provides methods for performing rationaldrug design to develop drugs that can modulate activity of γ-secretaseand thereby ameliorate a disorder, including but not limited to anAβ-related disorder. Such rational drug design can be performed usingcompounds that have been identified as inhibitors (or activators) ofγ-secretase as a starting point. Thus, the present invention providesscreens and assays to allow more specific inhibitors (or activators) tobe identified. Such methods of rational drug design are well-known inthe art. In a specific embodiment, the rational drug design methodsdisclosed in U.S. patent application Ser. No. 09/419,379, by Bibb etal., entitled “Methods of Identifying Agents That RegulatePhosphorylation/Dephosphorylation in Dopamine Signaling,” filed Oct. 15,1999, and Ser. No. 09/687,959, by Bibb et al., entitled “Methods ofIdentifying Agents That Regulate Phosphorylation/Dephosphorylation inDopamine Signaling,” filed Oct. 13, 2000, incorporated herein byreference in their entireties, are used.

Indeed, potential modulators can be examined through the use of computermodeling using a docking program such as GRAM, DOCK, or AUTODOCK(Dunbrack et al., 1997, Folding & Design 2:27-42), to identify compoundsas potential modulators of an ATP-dependent enzymatic activity, e.g.,ATP modulators such as those that modulate γ-secretase. These modulatorscan then be tested for their effect. In one embodiment, this procedurecan include computer fitting of potential modulators to the γ-secretasecomplex to ascertain how well the shape and the chemical structure ofthe potential modulator will bind to γ-secretase (see, e.g., Bugg etal., 1993, Scientific American 269(6):92-98; West et al., 1995, TIPS,16:67-74). Computer programs can also be employed to estimate theattraction, repulsion, and steric hindrance of the subunits with amodulator/inhibitor. Generally the tighter the fit, the lower the sterichindrances, and the greater the attractive forces, the more potent thepotential modulator since these properties are consistent with a tighterbinding constant. Furthermore, the more specificity in the design of apotential drug the more likely that the drug will not interact as wellwith other proteins. This will minimize potential side-effects due tounwanted interactions with other proteins.

Initially, in one embodiment, compounds known to bind to γ-secretase onany molecule that regulates γ-secretase activity or affects an enzymecomplex that contain γ-secretase can be systematically modified bycomputer modeling programs until one or more promising potential analogsare identified. In addition systematic modification of selected analogscan then be systematically modified by computer modeling programs untilone or more potential analogs are identified. Such analyses are wellknown to those of skill in the art and have been shown to be effectivein the development of, e.g., HIV protease inhibitors (see, e.g., Lam etal., 1994, Science 263:380-384; Wlodawer et al., 1993, Ann. Rev.Biochem. 62:543-585; Appelt, 1993, Perspectives in Drug Discovery andDesign 1:23-48; Erickson, 1993, Perspectives in Drug Discovery andDesign 1: 109-128).

Any of the potential agents or targets for the potential agents (e.g.,γ-secretase, Aβ, kinases, ATP) can be labeled. Suitable labels includeenzymes (e.g., alkaline phosphatase or horseradish peroxidase),fluorophores (e.g., fluorescein isothiocyanate (FITC), phycoerythrin(PE), Texas red (TR), rhodamine, free or chelated lanthamide seriessalts, especially Eu3+, to name a few fluorophores), chromophores,radioisotopes, chelating agents, dyes, colloidal gold, latex particles,ligands (e.g., biotin), chemiluminescent agents, magnetic beads ormagnetic resonance imaging labels. When a control marker is employed,the same or different labels may be used for the receptor and controlmarker.

In embodiments wherein a radioactive label, such as the isotopes ³H,¹⁴C, ³²P, ³⁵S, ³⁶Cl, ⁵¹Cr, ⁵⁷Co, ⁵⁸Co, ⁵⁹Fe, ⁹⁰Y, ¹²⁵I, ¹³¹I, and ¹⁸⁶Reis used, standard counting procedures known in the art may be utilized.

In embodiments wherein the label is an enzyme, detection may beaccomplished by any of the presently utilized colorimetric,spectrophotometric, fluorospectrophotometric, amperometric or gasometrictechniques known in the art.

A direct label is an example of a label that can be used according tothe methods of the present invention. A direct label is an entity that,in its natural state, is readily visible, either to the naked eye (forexample, by visual inspection through a compound or dissecting lightmicroscope), or with the aid of an optical filter and/or appliedstimulation, e.g., U.V. light to promote fluorescence. Examples ofcolored labels that can be used according to the methods of the presentinvention, include metallic sol particles, for example, gold solparticles such as those disclosed by Leuvering (U.S. Pat. No.4,313,734); dye sol particles such as disclosed by Gribnau et al. (U.S.Pat. No. 4,373,932) and May et al. (WO 88/08534); dyed latex such asdisclosed by May et al. (WO 88/08534), Snyder (EP-A 0280 559 and 0 281327); or dyes encapsulated in liposomes as disclosed by Campbell et al.(U.S. Pat. No. 4,703,017).

Other direct labels include a radionucleotide, a luminescent moiety, ora fluorescent moiety including, but not limited, to, e.g., amodified/fusion chimera of green fluorescent protein (as disclosed inU.S. Pat. No. 5,625,048, issued Apr. 29, 1997, and WO 97/26333,published Jul. 24, 1997, each of which is incorporated herein byreference in its entirety).

In addition to these direct labeling devices, indirect labels comprisingenzymes can also be used according to the present invention. Varioustypes of enzyme-linked immunoassays are well known in the art, forexample, enzyme-linked immunoassays using alkaline phosphatase,horseradish peroxidase, lysozyme, glucose-6-phosphate dehydrogenase,lactate dehydrogenase, or urease. These and other similar assays arewell known in the art and are disclosed, e.g., in Engvall (1980, “EnzymeImmunoassay ELISA and EMIT,” in Methods in Enzymology, 70: 419-439) andin U.S. Pat. No. 4,857,453.

In certain embodiments, proteins can be labeled by metabolic labeling.Metabolic labeling occurs during in vitro incubation of the cells thatexpress the protein in the presence of culture medium supplemented witha metabolic label, such as [³⁵S]-methionine or [³²P]-orthophosphate. Inaddition to metabolic (or biosynthetic) labeling with [Cl₂]-methionine,the invention further contemplates labeling with [¹⁴C]-amino acids and[³H]-amino acids (with the tritium substituted at non-labile positions)(see, e.g., U.S. patent application Ser. No. 09/419,379, by Bibb et al.,entitled “Methods of Identifying Agents That RegulatePhosphorylation/Dephosphorylation in Dopamine Signaling,” filed Oct. 15,1999, and Ser. No. 09/687,959, by Bibb et al., entitled “Methods ofIdentifying Agents That Regulate Phosphorylation/Dephosphorylation inDopamine Signaling,” filed Oct. 13, 2000, incorporated herein byreference in their entireties).

5.2.1. Assays for γ-Secretase Activity

According to the methods of the invention, modulators (e.g., inhibitorsor activators) of γ-secretase, can be detected and isolated usingmethods commonly known in the art, and/or identified by direct assay ofisolated enzyme. Such assays are well known to those of skill in theart.

In one embodiment, the methods of Roberts et al. (U.S. PatentApplication 20020025540, published Feb. 28, 2002, entitled “Isolation offunctionally active gamma-secretase protein complex and methods fordetection of activity and inhibitors thereof”) are used.

In another embodiment, the methods of Zhang et al. (2001, Biochemistry40: 5049-5055) are used.

In another embodiment, γ-secretase activity may be assayed according tothe methods known in the art, e.g., the methods of De Strooper et al.,1999, Nature 398: 518-522.; Hsiao et al., 1996, Science 274: 99-102.; Liet al., 2000, Proc. Natl. Acad. Sci. USA 97: 6138-6143.; Lichtenthaleret al., 1999, FEBS Lett. 453: 288-292; McLendon et al., 2000; FASEB J.14: 2383-2386; or Seiffert et al., 2000, Brain. Res. Mol. Brain Res. 84:115-126.

In another embodiment, γ-secretase activity may be assayed according tothe methods that assay for cleavage of proteins other than APP orAPP-like proteins. For example, proteolytic cleavage of the Notchprotein, which is a single transmembrane domain cell surface receptorthat mediates many cell fate decisions in vertebrates and invertebrates(Artavanis-Tsakonas et al., 1996, Science 268:225-232; Kopan and Turner,1996, Curr. Opin. Neurobiol. 6:594-601; Weinmaster, 1997, Mol. Cell.Neurosci. 9:91-102) may be assayed using the methods of Ray et al.(1999, J. Biol. Chem. 274:36801-36807) or Schroeter et al. (1998, Nature393:382-386), which are incorporated herein by reference in theirentireties.

γ-secretase may be immunoprecipitated from brain homogenate usingstandard methods, using, e.g., a commercial antibody directed againstγ-secretase.

In yet another embodiment, γ-secretase activity is assayed according tothe methods disclosed hereinbelow, e.g. in Example 4.

5.2.2. Assays for Aβ Levels

Aβ levels may be determined according to any method known in the art. Inone embodiment, Aβ levels are determined according to the methods of Xuet al. (1997, Proc. Natl. Acad. Sci. USA 94: 3748-3752).

In other embodiments, Aβ levels are determined according to the methodsdisclosed hereinbelow, e.g., in Examples 1, 2 or 3.

In another embodiment, Aβ peptide may be assayed for, e.g., in a brainhomogenate, using a sandwich ELISA (e.g., Biosource International,Camarillo, Calif.) according the manufacturer's recommendations.

5.2.3. Animal Models

In vivo animal models of APP processing into Aβ and of Aβ-relateddisorders such as Alzheimer's disease are well known to those of skillin the art. Such animal models can, for example, be used to furtherscreen compounds identified via the methods presented herein.Alternatively, such models can be used to routinely validate compoundsfor efficacy in use in the modulation, prevention, treatment, andsymptom amelioration methods of the present invention.

In one embodiment, the wild-type guinea pig is used as an animal model.For example, wild-type guinea pigs may be used as models for APPprocessing into Aβ, for Aβ production and/or accumulation of Aβ inbrain. They are known to produce Aβ peptides that are immunologicallyidentical to human Aβ. Therefore, the guinea pigs provide, for example,a model in which to examine perturbations in APP metabolism with andwithout drug treatment.

In other embodiments, a mouse model of Alzheimer's disease is used,e.g., a transgenic mouse expressing human APP-Swedish (see, e.g.,Borchelt et al., 1996, Neuron 17:1005-1013), which exhibitsneuropathological symptoms similar to those of Alzheimer's diseasepatients. Moreover, a rat model of Alzheimer's disease can be used. Inanother embodiment, a transgenic mouse model of Alzheimer's disease canbe used, e.g., the Tg2576 transgenic mouse (U.S. Pat. No. 5,877,399).Such an animal model may be used to screen for compounds useful in thetreatment of Alzheimer's disease due to its exhibition of, e.g., analtered level of human Aβ.

Models such as these can also be used to assess the efficacy of anypotential therapeutic agents as disclosed hereinbelow. Generally, atleast two groups of animals are used in the assay, with at least onegroup being a control group in which the administration vehicle isadministered without the potential therapeutic agent.

According to the invention, animal models may be employed that have, incertain embodiments, an altered physiological regulation of the nervoussystem such that the animal or tissues derived from it can be utilizedfor screening of potential therapeutic agents and/or therapeuticregimens that act at the level of γ-secretase activity, especially atthe level of competing (directly or indirectly) with ATP for activationof γ-secretase. Drugs that can reverse any of the defects exhibited bysuch an animal model act at some point in the catabolic pathwayunderlying the cleavage of APP to Aβ, and are thus of potential usetherapeutically. Additionally, since some defects occur at thebehavioral level, the alteration or modulation of these behavioraldefects can have a high predictive value for therapeutic use inmodification of such behaviors.

According to the methods of the invention, animal models thus can beused, as screening tools to elucidate the mechanisms of APP cleavage toAβ involved in both normal and diseased patient populations. Forexample, an animal model can thus be utilized to assess the response toa variety of potential therapeutic strategies and therapeutic agentsthat can thus be used in the treatment of patients suffering from avariety of neurological diseases and disturbances.

Using animal models, various small molecule drugs can be screened forpotentially advantageous effects, including enhanced potency as well asminimization of side effects. Typical candidates for such screening maybe obtained from any of several commercially available drug libraries.

Specific disorders for which an animal model can be utilized include,but are not limited to, an Aβ-related disorders, as disclosed herein. Bymeasuring various characteristics in an animal model in response toadministration of endogenous or exogenous agents, and comparing thesecharacteristics to those in an animal treated with a potentialtherapeutic agent, an assessment of the utility of the potentialtherapeutic agent in the prevention and/or treatment of a particulardisorder or disease state can be made. For instance, the potentialtherapeutic agent can be administered to an animal model for aparticular Aβ-related disorder, and the Aβ level in a control animal(e.g., an animal administered vehicle) can then provide an indication ofthe value of the potential therapeutic agent.

Another aspect of the invention is a method for selecting a therapeuticagent for possible use in the treatment of a disorder, including but notlimited to, an Aβ-related disorder, which comprises administering asuspected therapeutic agent to an animal model, and measuring and/ordetermining the putative therapeutic agent's effect on any phenotypiccharacteristics which may be believed to be related to said disorder.

In one embodiment of this aspect of the invention, a suspectedtherapeutic agent is administered to an animal model, e.g., an animalmodel for an Aβ-related disorder, and the Aβ level is measured for theanimal model, wherein the normal Aβ level of the animal model in theabsence of a therapeutic agent is characteristically different than thatof control (e.g., wild-type) animals. The potential therapeutic agentsare selected on the basis of whether there is a statistical significancebetween a test characteristic, e.g., a test Aβ level and a normalcharacteristic, e.g., a normal Aβ level. Potential therapeutic agentsare selected that show a statistically significant change in thecharacteristic that is measured or determined. In a preferredembodiment, the test characteristic of the animal model in the absenceof a therapeutic agent is different than that of control (e.g.,wild-type) animals to which the potential therapeutic has not beenadministered.

Yet another aspect of the present invention is a method for selecting atherapeutic agent for possible use in the treatment of Alzheimer'sdisease which comprises administering a suspected therapeutic agent toan animal model, e.g., an animal model for Alzheimer's disease, andmeasuring and/or determining the putative therapeutic agent's effect onany of the phenotypic characteristics which may be believed to berelated to Alzheimer's disease.

The following experimental examples are offered by way of illustrationand not by way of limitation.

6. EXAMPLE 1 Production of Aβ is ATP-Dependent

The working example presented herein demonstrates that production of Aβis ATP-dependent, and presents data indicating that p-secretase activityis at least partially ATP-independent, while optimal γ-secretaseactivity requires ATP.

6.1. Materials and Methods

Whole N2a cells doubly transfected with the human Swedish APP mutationand the presenilin-1 (PS 1) mutant, ΔE9, were ⁵S-pulse-labeled beforepermeabilization and chased at 20° C. to localize newly synthesized,³⁵S-labeled APP to the Golgi apparatus, a major location for APP and thepredominant site of Aβ production. Cells were then permeabilizedaccording to the methods of Xu et al. (1997, Proc. Natl. Acad. Sci. USA94: 3748-3752), depleted of cytosol and incubated at 37° C. either withan ATP regenerating system or with apyrase to hydrolyze residual ATP andother nucleotide triphosphates, and Aβ production was assayed. Theeffect of adding an ATP-regenerating system to a cell-free assay systemfor Aβ generation was quantitated according to the methods of Xu et al.(1997, Proc. Natl. Acad. Sci. USA 94: 3748-3752).

For the assay of isolated γ-secretase activity, membranes were isolatedfrom N2a cells expressing a recombinant γ-secretase substrate, C99,according to the methods of Zhang et al. (2001, Biochemistry 40:5049-5055). Accumulation of human C99 (also known as β C-terminalfragment or βCTF) in this system results from expression of thetransgene and not from endogenous BACE (β-secretase) cleavage of mouseAPP; furthermore, the use of human specific anti-Aβ antibody does noteffectively resolve the small amount of endogenous mouse Aβ producedunder the experimental conditions, thus, allowing measurement of only Aβproduced from the action of γ-secretase on the human C99. Also, todetermine whether ATP was primarily responsible for stimulatingγ-secretase activity, the previous energy regenerating system wassubstituted with one designed to sustain concentrations of ATP alone butnot GTP. Aβ release from isolated membranes was determined by ELISA(Biosource International, Camarillo, Calif.).

6.2. Results

Studies were performed to examine possible energy requirements of Aβproduction. Aβ production was reconstituted in a cell-free systemconsisting of permeabilized cells, the cytosol of which had been washedaway, leaving intracellular membranes and organelles intact.

To identify steps in the APP processing pathway that might require ATP,accumulation of APP metabolites was studied using ³⁵S pulse labeling ina cell-free system consisting of N2a cells doubly transfected with thehuman Swedish APP mutation and the presenilin-1 (PS 1) mutant, ΔE9. Bothmutations result in familial forms of Alzheimer's disease characterizedby a higher Aβ42/Aβ40 ratio (Borchelt et al., 1996, Neuron17:1005-1013). This cell line has been shown to produce large amounts ofAβ peptides and of C99, a product of β-secretase cleavage of APP and animmediate precursor of Aβ. The responses of both β-secretase andγ-secretase activities to ATP depletion were measured and compared.

ATP stimulated Aβ production above the initial level of Aβ that hadaccumulated during the 20° C. chase. The results of these experiments,showing radiolabeled Aβ production from precursor accumulated in theGolgi apparatus, are shown in FIG. 1. In particular, the energyregenerating system resulted in a three-fold increase in Aβ productioncompared to apyrase treated samples (FIG. 1) and concomitantly, atwo-thirds decrease in C99 (also known as β C-terminal fragment or βCTF)(FIG. 2).

A much smaller quantity of Aβ was produced in the absence of ATP,demonstrating that production of Aβ is ATP-dependent.

By contrast, C99 production from APP was increased in the absence ofATP, indicating that the activity of β-secretase is at least partiallyATP-independent. This result also indicates that the increase in C99likely results from a decrease in γ-secretase activity, that allows foraccumulation of the Aβ precursor. This is evident in the autoradiogramshown in FIG. 2.

Virtually, identical results were achieved when apyrase was left out andsamples were incubated in the absence of the energy regenerating system(data not shown).

These results indicate that, in the context of Aβ production, optimalγ-secretase activity requires nucleotide triphosphates and thatsubstantial β site APP cleaving enzyme (also known as β-secretase or“BACE”) activity still occurs in their absence.

In order to test whether isolated γ-secretase activity is ATP-dependent,an assay of isolated γ-secretase activity was performed. Generation ofAβ in the isolated γ-secretase assay was strongly stimulated by additionof ATP and a regenerating system (Xu et al., 1997, Proc. Natl. Acad.Sci. USA 94: 3748-3752) (FIG. 3). A dose-dependent decrease in Aβgeneration was observed upon addition of Compound 1 (see below) to thesemembranes (FIG. 4).

In summary, the results presented herein demonstrate that production ofAβ is ATP-dependent. The results further indicate that β-secretaseactivity is at least partially ATP-independent, and optimal γ-secretaseactivity requires ATP.

7. EXAMPLE 2 Modulation of Aβ Levels

The working example presented herein demonstrates the successful use ofcompounds to modulate, in particular lower, Aβ levels.

7.1. Materials and Methods

N2a cells transfected with the human Swedish APP mutation, and witheither normal human presenilin 1 or ΔE9 mutant presenilin 1 (PS1), wereused for assays of compound effect on Aβ generation.

Media was added with compound concentration of 0, 10⁻⁹, 10⁻⁸, 10⁻⁷ and10⁻⁶ M; cells were incubated overnight at 37° C. The next day, cellswere incubated with ³⁵S-methionine and ³⁵S-cysteine for four hours inthe presence of the compound as above.

Media and cells were separately harvested and subjected toimmunoprecipitation with antibody 4G8 (Senetek, Napa, Calif.), whichrecognizes an epitope corresponding to residues 17-28 of Aβ. Antibody4G8 immunoprecipitates Aβ from media, and Aβ plus full-length APP fromcells. After removal of antibody 4G8 and bound Aβ from media, thesupernatant was reacted with 22C11 (Chemicon International), an antibodythat immunoprecipitates sAPPβ and sAPPα. Immunoprecipitates weresubjected to PAGE and resolved by autoradiography according to standardmethods. APP metabolites were compared by densitometry according tostandard methods.

In other experiments, immunoprecipitation followed by Western blotanalysis was carried out on unlabeled (no radioactive label) N2a cells,using standard methods and using 6E10 (Senetek, Napa, Calif.) or 22C11antibodies for detection.

Mass spectrometry was carried out on media from N2a cells to detect Aβ40and Aβ42 peptides using the CiphergenProteinChip System (CiphergenBiosystems Inc., Fremont, Calif.), which is based on Seldi andtime-of-flight mass spectrometry, using standard protocols as set out bythe manufacturer. The experimental procedures were the same as thosedisclosed above for immunoprecipitation/Western blotting, withoutmetabolic labeling. Unlike immunoprecipitation/Western blotting, mediadid not contain sodium lauryl sulfate (SDS) but did contain Triton X-100detergent. Aliquots of detergent treated media were contacted to a metalchip that was previously contacted with an antibody, such as 6E10. Theantibody binds human APP metabolites, including Aβ, in the appliedsample. Detection of bound antigen followed a laser pulse thatdissociated antigen from antibody.

Aβ peptides were resolved graphically as peaks of various heights,situated on an x-axis denoting molecular mass. Thus, Aβ40 and Aβ42 wereresolved separately at approximately 4333 and 4519 daltons,respectively. The relative heights or areas under the peaks in a givenspectrum denoted the relative concentrations of the detected species.Peaks corresponding to the same species, as conveyed by molecular mass,were compared among different spectra, reflecting different samples, tomeasure relative quantities of Aβ among a set of samples.

These immunoprecipitation experiments were repeated using a minimaltreatment time, 1 hour instead of overnight.

7.2. Results

Compounds were tested for an ability to modulate Aβ levels. Inparticular, STI-571, a member of the phenyl aminopyrimidine class ofpharmacophores was tested. Specifically, the mesylate salt of STI-571(also known as GLEEVEC™, Novartis; see FIG. 20) was tested. STI-571 isan ATP competitor known to target the ATP binding site on BCR-Abltyrosine kinase, and also to inhibit Abl, c-kit and platelet-derivedgrowth factor receptor (PDGFR). An STI-571 variant, WGB-BC-15 (see FIG.20), was also tested. In addition, Compounds 1 and 2 (see FIG. 20),members of the pyrimidinyl pyridone tyrosine kinase inhibitor class ofmolecules, were tested.

At concentrations in the range of 1×10⁻⁶ M, STI-571 exhibited an IC50(that is, inhibited total levels of Aβ by 50%) in N2a cells expressingAPP and either normal human presenilin 1 or ΔE9 mutant presenilin 1.WGB-BC-15 also inhibited levels of secreted Aβ. Compound 1 inhibitedtotal Aβ levels as well, with an IC50 of less than 10⁻⁶ M.

Intracellular levels of Aβ also decreased in the presence of thecompounds. These results were derived from immunoprecipitation/Westernblot analysis and corroborated by results of studies on APPincorporation of ³⁵S-methionine.

Levels of sAPPα and sAPPβ, two of the major secreted metabolites of APP,were not inhibited by these compounds. Levels of sAPPα increased athigher concentrations of the compounds. These results demonstrate thatthe reduction observed in secreted Aβ levels was not due to somenon-specific deleterious affect on the cells' protein secretion ability.

Mass spectrometry was carried out according to standard methods, bywhich Aβ40 and Aβ42 were clearly resolved, revealing that levels of bothspecies were dramatically reduced in the presence of Compound 1 (FIG.5).

FIGS. 6 and 7 illustrate the dose-dependent decrease in Aβ observedafter just one hour of treatment of ³⁵S labeled N2a cells expressing thehuman APP-Swedish transgene with STI-571 or Compound 1, respectively.

8. EXAMPLE 3 Modulation of Aβ Levels in Primary Neuronal Cell Cultures

This example demonstrates that STI-571, Compound 1 and Compound 2inhibit production of Aβ peptides in normal rat primary neuronal cellcultures. These result correlate well with the in vivo results describedbelow (see Section 9).

8.1. Materials and Methods

Cultures of rat embryonic cortical neurons were established from day 18pregnant SD rats using standard techniques (Gasparini et al., 2001, J.Neurosci. 21(8):2561-2570). After several days of culture, new media wasadded along with STI-571, Compound 1 or Compound 2 at concentrations of0, 10⁻⁹, 10⁻⁸, 10⁻⁷ 2×10⁻⁶ and 10⁻⁶ M; cells were incubated overnight at37° C.

The next day, cells were labeled with ³⁵S-methionine and cysteine forfour hours in the presence of compounds as above. Media and cells werethen harvested and subjected to immunoprecipitation with antibody 4G8,to detect Aβ and APP. The media was serially immunoprecipitated firstwith antibody 4G8 and then antibody 22C11, for detection of secreted Aβ,sAPPP and sAPPα. Immunoprecipitates were subjected to PAGE and resolvedby autoradiography; APP metabolites were compared by densitometry.

8.2. Results

Results similar to those obtained in Example 2 were obtained in theseexperiments with rat cortical neurons exposed to Compound 1. Compound 1reduced levels of Aβ in both media (FIG. 8A) and cells. The predominantAβ peptide produced by the neurons was Aβ11-40, which is characteristicof rat primary neurons (FIG. 8B). Total sAPP was not reducedsignificantly nor was total cellular APP (FIG. 9), indicating that thecompound was not cytotoxic nor did it non-specifically inhibit cellularsecretion. Similar results were obtained with related Compound 2 (FIG.10).

The time course and dose response of primary cortical neuronal culturesto STI-571 and Compound 1 were studied in more detail in a second set ofexperiments similar to those described above. Significant reductions inAβ were observed in as little as 6 hours (FIGS. 11 and 12) and 5 μMSTI-571 (FIG. 13) or 0.5 μM Compound 1 (FIG. 14).

9. EXAMPLE 4 Modulation of Aβ Levels in an Animal Model of Aβ Production

The working example presented herein demonstrates the successfulreduction of levels of brain Aβ40 and Aβ42 in wild-type guinea pigs, ananimal model of Aβ production. Wild-type guinea pigs are an art-acceptedmodel for normal Aβ production and are known to produce Aβ peptides thatare immunologically identical to human Aβ. Therefore, wild-type guineapigs provide an animal model for examining perturbations in APPmetabolism with and without drug treatment, and, as such also representan animal model for a physiological hallmark of Alzheimer's disease.

9.1. Materials and Methods

Osmotic minipumps containing 200 μl compound STI-571 (50 mM in salinebuffer, final concentration of 15.4 mg/kg) were implanted intrathecallyin 300-350 g guinea pigs. Two hundred microliters of Compound 1 (10 mM,final 2.8 mg/kg) in 100% DMSO was placed in a small catheter attached tothe minipump.

Compounds were tested for an ability to modulate Aβ levels. Inparticular, STI-571, a member of the phenyl aminopyrimidine class ofpharmacophores was tested. Specifically, the mesylate salt of STI-571(also known as GLEEVEC™, Novartis) was tested. STI-571 is an ATPcompetitor known to target the ATP binding site on BCR-Abl tyrosinekinase, and also to inhibit Abl, c-kit and platelet-derived growthfactor receptor (PDGFR). An STI-571 variant, WGB-BC-15 (see FIG. 20),was also tested. In addition, Compounds 1 and 2 (see FIG. 20), membersof the pyrimidinyl pyridone tyrosine kinase inhibitor class ofmolecules, were tested.

After 7 days of treatment, animals were sacrificed, and their brainshomogenized in 25 mM Tris 7.5, 50 mM NaCl, 1 mM DTT, 5 mM EDTA, 1 mMEGTA, and 1× protease inhibitor cocktail (Complete™, BoehringerBiochemicals). Aliquots of brain homogenate were assayed for Aβ peptideby using a sandwich ELISA according the manufacturer's recommendations(Biosource International, Camarillo, Calif.) and for C99 byimmunoprecipitation and Western blot analysis using standard methods.

9.2. Results

Levels of brain Aβ40 and Aβ42 were reduced (FIGS. 15A and 15B), and C99increased (FIGS. 15C and 15D), in animals treated with 15.4 mg/kgSTI-571. Similar changes were seen with 2.8 mg/kg Compound 1 (FIGS.16A-D). Animals appeared healthy and active at these doses of compound.

These data demonstrate that in vivo, STI-571 and Compound 1 inhibitproduction of Aβ peptides. Further, because the amounts of C99 rosesignificantly, the compounds represent putative γ-secretase inhibitors,as the reduction in Aβ levels is likely due to the specific cleavage ofC99.

10. EXAMPLE 5 Administration of STI-571 or Compound 1 Does not Inhibitthe Cleavage of Notch

A major concern related to the use of γ-secretase inhibitors is theirpotential for inhibition of cell signaling through the Notch cellsurface receptor. Notch receptors function in a variety of cell fatedecisions in the developing nervous system and function in adult life inthe immune system. Thus, it is generally preferred that the Aβinhibition not significantly inhibit cleavage of Notch. This workingexample demonstrates that administration of STI-571 or Compound 1 tocultured cells at levels that strongly inhibit Aβ formation does notaffect Notch cleavage. As such, these compounds represent selectiveinhibitors of γ-secretase activity.

10.1. Materials and Methods

N2a cells were transfected with a constitutively active truncated formof Notch, mouse “deleted-extracellular” Notch (mΔe-Notch), whichsupplies a direct substrate for γ-secretase (De Strooper et al., 1999,Nature 398: 518-522).

10.2. Results

At levels of STI-571 (FIG. 17) or Compound 1 (FIG. 18) that stronglyinhibit Aβ-formation, cleavage of mΔe-Notch to form Notch intracellulardomain (NICD) was unaffected.

11. EXAMPLE 6 STI-571 and Compound 1 Inhibit Production of Aβ byInhibition of ARG Kinase but not ABL Kinase

Abl tyrosine kinases are known targets of STI-571 (Okuda et al., 2001,Blood 97(8): 2440-2448). Abl kinase is a known target of Compound 1. Todetermine whether Abl kinase is required for Aβ production and whetherthe effects of the inhibitors STI-571 and Compound 1 on Aβ productionresult from inhibition of Abl kinase, the experiments described hereinwere conducted. The results presented in this working exampledemonstrate that STI-571 and Compound 1 inhibit production of Aβ whetheror not Abl kinase is present.

11.1. Materials and Methods

Abl knockout (Abl−/−) 3T3 mouse fibroblasts and wild-type 3T3 mousefibroblasts (see Liu et al., 1996, Nature 384(6606):273-6) were grownseparately and to confluence in 100 mm Corning tissue culture plates.These fibroblast cell lines are permanent cell lines. Several plateswere exposed to either 5 μM or 10 μM STI-571 dissolved in water, or tono STI-571. Several other plates were exposed to either 0.5 μM or 1.0 μMCompound 1 (dissolved in DMSO) or to DMSO without Compound 1.

For the above-described cell cultures, each of the drugs or controlsolvents was added to a standard culture medium (DMEM+10% FBS+pen/strep)at a dilution of 1:1000 and cultures were then incubated at 37° C. for 2hours. The contacting of cell cultures and incubation with inhibitorsand solvent, as described above, constituted “pretreatment” of cells.

The first culture medium was then removed and the cell cultures wererinsed with a second culture medium lacking methionine, cysteine andglutamate. The cell cultures were incubated with this culture medium for30 minutes (to stimulate subsequent uptake of amino acids). Then thesecond culture medium was exchanged for fresh first culture mediumcontaining ³⁵S-labeled methionine and cysteine (NEN Express label 50μl/ml) and unlabeled glutamine (final concentration 2 mM) and withinhibitors as described above, and incubated for 4 hours at 37° C.

Media was collected and immunoprecipitated with 4G8 mouse monoclonalantibody (Senetek, Napa, Calif.) to detect Aβ peptides and resolved byPAGE and autoradiography, according to standard methods (as describedhereinabove). Cells were dissolved in 3% SDS, diluted inimmunoprecipitation buffer containing 1% Triton X-100 andimmunoprecipitated with 369 rabbit polyclonal antiserum (producedaccording to standard methods) to detect APP and resolved by PAGE andautoradiography.

11.2. Results

Both wild-type 3T3 fibroblasts and Abl −/−3T3 fibroblasts producedsimilar quantities of Aβ peptide and p3 peptide (both products ofγ-secretase activity) compared to the quantities of APP produced by eachcell line (quantities of APP were also similar in both cell lines). Thisdemonstrates that Abl kinase is not necessary for Aβ production or forγ-secretase activity.

Furthermore, both STI-571 and Compound 1 inhibited Aβ production in boththe 3T3 fibroblast and Abl −/−3T3 fibroblast cells lines to similardegrees, and this inhibition was also similar to the degree ofinhibition of Aβ production achieved by the inhibitors in N2a cells.This indicates that the target of the inhibitors, responsible for Aβinhibition, was intact, whether or not Abl kinase was present.

12. EXAMPLE 7 Mechanism of Action of γ-Secretase Activity

It was previously noted (in FIGS. 17 and 18) that Notch-I cleavage isnot inhibited by the inhibitors STI-571 and Compound 1. Significantly,Notch cleavage is known to be catalyzed by γ-secretase activity. Thusγ-secretase inhibitors that inhibit Aβ production but not Notch cleavage(i.e., selective γ-secretase inhibitors) may be used as therapeuticagents because they potentially produce fewer side effects that couldinjure the immune system, as well as other Notch dependent systems.

This working example demonstrates that Notch-1 cleavage is not energy orATP-dependent, or differentially affected by the presence (versus theabsence) of ATP. Thus, without wishing to be bound by any particulartheory, ATP modulators, such as inhibitors that compete with ATP forbinding to protein targets, do not inhibit Notch cleavage and will notbe cytotoxic or affect cellular or developmental processes regulated bythe Notch protein.

12.1. Materials and Methods

A cell-free system derived from mΔe-Notch 1 transfected N2a cells (Leemet al., 2002, Neurobiol Dis. 11(1):64-82) was created according to themethods described in Example 1 (see also, Xu et al., 1997, Proc. Natl.Acad. Sci. USA 94: 3748-3752). The cells were pulse-labeled withradioactivity and chased at 20° C. for 2 hours to accumulate labeledNotch in the trans Golgi network (TGN). The cells were then incubated at37° C. for 20 to 25 minutes to synchronize localization of mΔe-Notch 1to the cell surface (considered the primary site for Notch 1 cleavage).The cells were then permeabilized and incubated either with or without astandard ATP energy regenerating system with GTP (Xu et al., 1997, Proc.Natl. Acad. Sci. USA 94: 3748-3752). Samples were incubated, under thesame conditions, either with or without a standard γ-secretase inhibitor(Calbiochem).

To measure mΔe Notch-1 and Notch intracellular domain (NICD, the productof Notch cleavage), permeabilized cells were sedimented and bothsedimented cells and supernatant were immunoprecipitated with anti-mycantibody according to standard methods to detect Notch and NICD (bothwere labeled with myc tags).

12.2. Results

NICD was found in the supernatant because the cells are permeabilizedand hence NICD, which would otherwise be in the cytosol, could beretrieved by sedimenting the permeabilized cells, thus separating themfrom the supernatant. Furthermore, the standard γ-secretase inhibitorinhibited Notch cleavage as expected (FIG. 19), showing that γ-secretaseactivity is necessary for Notch cleavage in this system.

It was previously shown, in Section 10, that at levels of STI-571 (FIG.17) or Compound 1 (FIG. 18) that strongly inhibit Aβformation, cleavageof mΔe-Notch to form Notch intracellular domain (NICD) was unaffected.In the present example, it was demonstrated that Notch is cleavedequally effectively in the presence or absence of a standard γ-secretaseinhibitor, and in the presence or absence of ATP. Thus Notch cleavage isnot ATP-dependent.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description. Suchmodifications are intended to fall within the scope of the appendedclaims.

All references cited herein are incorporated herein by reference intheir entireties and for all purposes to the same extent as if eachindividual publication, patent or patent application was specificallyand individually indicated to be incorporated by reference in itsentirety for all purposes.

The citation of any publication is for its disclosure prior to thefiling date and should not be construed as an admission that the presentinvention is not entitled to antedate such publication by virtue ofprior invention.

1. A method for reducing amyloid-β peptide (Aβ) plaque in a cell ortissue which produces Aβ plaque, comprising contacting said cell ortissue with an amount of a compound sufficient to decrease Aβ levels insaid cell or tissue, wherein said compound modulates an ATP-dependentenzymatic activity wherein said compound is WGB-BC-15, as illustratedbelow

or a pharmaceutically acceptable salt thereof.
 2. The method of claim 1wherein the Aβ plaque is neuritic plaque.
 3. The method of claim 1wherein the Aβ comprises metabolites of β-amyloid precursor protein. 4.The method of claim 1 wherein the Aβ is Aβ42.
 5. The method of claim 1wherein the compound increases levels of sAPPα.
 6. The method of claim 1wherein the compound does not inhibit Notch-1 cleavage.
 7. The method ofclaim 1 wherein the enzymatic activity is a γ-secretase activity.
 8. Themethod of claim 1 wherein the enzymatic activity is a kinase activity.9. The method of claim 8 wherein the kinase is a tyrosine kinase. 10.The method of claim 9 wherein the tyrosine kinase is Abl kinase, BCR-Ablkinase, ARG kinase, src kinase, c-kit or platelet-derived growth factorreceptor.
 11. The method of claim 9 wherein the kinase is aserine/threonine kinase, a carbohydrate kinase or a lipid kinase.
 12. Amethod for reducing Aβ plaque levels in a cell or tissue which producesAβ plaque, comprising contacting said cell or tissue with an amount ofan ATP modulator sufficient to decrease Aβ levels in said cell or tissuewherein said compound is WGB-BC-15, as illustrated below

or a pharmaceutically acceptable salt thereof.